Uses of parasite macrophage migration inhibitory factors

ABSTRACT

This invention relates to compositions (e.g. vaccine compositions) which can be used to provide a subject with protective immunity against a parasite infection. The compositions comprise: (i) an immunologically effective amount of a nucleic acid (e.g. a nucleic acid-based vaccine) comprising a sequence which encodes a parasite macrophage migration inhibitory factor (MIF) antigen; (ii) a parasite MIF antigen; or (iii) an antibody which specifically binds to a parasite MIF antigen. The compositions may be used to treat infections and diseases caused by parasitic protozoans, such as a  Plasmodium  parasite, or parasitic helminths.

CROSS-REFERENCED TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/128,851, with a 371(c) date of Sep. 23, 2016, which is the U.S.national phase of PCT/EP2015/056310, filed Mar. 24, 2015, which claimsthe benefit of European patent application 14161614.4, filed Mar. 25,2014, the disclosures of which are herein incorporated by reference intheir entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 303822019001SEQLIST.TXT,date recorded: Oct. 16, 2020, size: 19 KB).

TECHNICAL FIELD

This invention is in the field of treating and preventing parasiteinfections. In particular, the present invention relates to the use ofparasite macrophage migration inhibitory factors (MIFs) for preventingparasite infections such as malaria.

BACKGROUND ART

Parasitic diseases caused by protozoa and helminths affect billions ofindividuals worldwide and cause millions of human deaths annually,particularly in tropical countries. Malaria, for example, which iscaused by Plasmodium protozoa, infects 300-500 million individualsannually and leads to more than 1 million deaths. More than a third ofthe global population is at risk of malaria. Disease mortality isprimarily caused by complications due to severe anemia, shock andcerebral malaria, which can be associated with an excessiveproinflammatory response. Malaria preferentially kills theimmunologically naïve, for example, young children. Recurrent orpersistent malaria infection can lead to “tolerance” to severe diseasebut memory CD4 T cells do not appear to be adequately maintained aftermalaria infection and fully protective and “sterilizing” immunity neverdevelops [1]. This inability to develop or maintain effective“sterilizing” immunity following infection has been recognised as acharacteristic of many other parasite infections in addition to malaria[2]. This makes vaccine development especially difficult.

The Leishmania parasite, a flagellated protozoan, is another major causeof parasitic disease. Leishmaniasis affects about 12 million individualsper year and leads to about 60,000 deaths, with about 350 millionthought to be at risk. Schistosomiasis is caused by parasitic helminthsof the genus Schistosoma and is thought to affect 200 million peopleworldwide and lead to about 20,000 deaths. Hookworm infection by thenematodes Necator americanus and Ancylostoma duodenale is estimated toaffect over 700 million individuals. Other parasitic diseases, such astoxoplasmosis, lymphatic filariasis, onchocerciasis, and Guinea wormdisease are thought to affect more than 1 billion people worldwidebetween them. However, researchers have struggled to develop vaccinesagainst such parasites due to their complex multi-stage life cycles,antigenic variability and immune evasion. Thus, there is still hugedemand for effective treatments which protect against parasiticinfections, such as malaria.

DISCLOSURE OF THE INVENTION

The present inventors unexpectedly found that macrophage migrationinhibitory factor (MIF) from a parasite can be used as an effectivevaccine antigen to provide protective immunity against parasiteinfection.

In particular, the inventors found that immunization of mice with aself-replicating RNA vaccine encoding Plasmodium berghei MIF ortholog(PbMIF) led to a measurable and significant decrease in parasitemiafollowing initial Plasmodium challenge and a pronounced reduction inparasitemia following cure and re-challenge. The reduced parasitemia wasaccompanied by an expansion of the Plasmodium-responsive memory T cellpopulation in the treated mice. The inventors also showed thatimmunization with an adjuvanted PbMIF antigen was well tolerated andinduced a robust anti-PbMIF immune response. Immunization with PbMIFtherefore allows for the increased development of memory T cells andprovides significant protection against malaria re-infection. Inaddition, the inventors showed that passive transfer of an anti-PbMIFantibody significantly reduced parasitemia following P. bergheiinfection.

In mammals, MIF is a ubiquitous and highly conserved proinflammatorycytokine which exhibits tautomerase and oxidoreductase enzymaticactivities and is involved in the regulation of a broad spectrum ofimmune responses. The role of MIF in the mammalian immune system hasbeen widely studied and it has been implicated in the pathogenesis ofseveral diseases such as septic shock, asthma, rheumatoid arthritis andinflammatory bowel disease [3,4]. The precise molecular mechanisms bywhich MIF functions are not yet well understood, but mammalian MIF hasbeen shown to bind and exert its inflammatory effects via the cellsurface receptor CD74 (also known as the MIF receptor, MIF-R)[5,6].However, the role of MIF was widely thought to be confined to the innateimmune system.

MIF orthologs or homologs are found in many parasitic organisms thatinfect mammals, including unicellular protozoan parasites such asPlasmodium, Leishmania and Toxoplasma and parasitic helminths andnematodes such as Brugia and Ancyclostoma. Despite often sharing onlylow levels of sequence identity, these parasite orthologs share closestructural and functional similarities with MIFs from their mammalianhosts [3,4,5]. Thus, the present invention is applicable to a wide rangeof parasites which express a MIF ortholog or homolog. For example, a MIFortholog produced by Leishmania major, has been identified which sharessignificant structural and functional homology with human MIF, includingtautomerase, chemotactic and anti-apoptotic activities and MIF-R binding[7]. MIF proteins produced by Plasmodium falciparum and Plasmodiumberghei have been shown to be similar to one another and to mammalianand other parasite MIFs, to interact with MIF-R and to share similarenzymatic and pro-inflammatory function.

In view of their structural conservation and distribution amongevolutionarily distant species, the present inventors hypothesised thatparasite MIF orthologs play a role in evasion of the host immuneresponse. Sun et al. [5] recently showed that Plasmodium MIF enhancedinflammatory cytokine production and induced activated CD4 T cells todevelop into short-lived effector cells rather than memory precursorcells in infected mice, preventing the establishment of immunologicalmemory. Also, CD4 T cells were more susceptible to apoptosis and CD4 Tcell recall responses against challenge infections were reduced.Speculative therapeutic applications targeting MIF have been proposed.For example, Dobson et al. [4] suggested that Plasmodium MIF could be apotential drug target and that it would be important to selectivelytarget parasite MIF relative to host protein. Vermeire et al. [3]suggested that drugs or vaccines specifically targeting nematode MIForthologs could have therapeutic value. Cho et al. [8] found thatimmunization of hamsters with a MIF ortholog from the hookwormAncylostoma ceylanicum alleviated clinical symptoms ofhookworm-associated disease (weight loss and anemia) and suggestedtargeting the hookworm MIF with small molecule inhibitors to treatinfection. However, none of these documents elucidated a precise rolefor MIF orthologs in parasitic infections and, prior to the presentinvention, no specific therapeutic applications of invertebrate parasiteMIF had been provided.

In contrast, the inventors have identified for the first time that animmune response against MIF can be used to provide protective immunityagainst a parasite infection. In particular, the inventors have shownthat parasite MIFs are viable vaccine candidates that may be used eitheras stand-alone antigens or in combination with other parasite antigensin order to promote long-lasting memory T cell responses and protectiveimmunity against parasite infection. The inventors' findings alsoestablish that an antibody-mediated immune response against parasite MIFcan usefully protect a subject against parasite infection. Protectiveimmunity against parasite infection may therefore be established in asubject by eliciting an immune response against parasite MIF and/orblocking parasite MIF function in the subject, thus enabling a subjectto develop protective immunological memory against the parasite,particularly when the subject is, or has been exposed to parasiteantigens other than MIF as well (e.g. due to infection or exposure toother parasite vaccines). Accordingly, in one aspect, the inventionprovides a method for providing protective immunity against a parasiteinfection in a subject in need thereof, comprising administering animmunologically effective amount of a composition to the subject,wherein the composition comprises: (i) a nucleic acid comprising asequence which encodes a parasite MIF antigen; (ii) a parasite MIFantigen; or (iii) an antibody which specifically binds to a parasite MIFantigen. In some embodiments, the method may comprise administering acombination of (i), (ii) and/or (iii).

In another aspect, the invention provides a composition for use in amethod of providing protective immunity against a parasite infection ina subject in need thereof, which comprises an immunologically effectiveamount of: (i) a nucleic acid comprising a sequence which encodes aparasite MIF antigen; (ii) a parasite MIF antigen; or (iii) an antibodywhich specifically binds to a parasite MIF antigen. The composition maybe a pharmaceutical composition. Accordingly, the composition may alsocomprise a pharmaceutically acceptable carrier. In certain embodiments,the composition of (i) or (ii) is a vaccine composition.

The invention also provides a method for providing protective immunityagainst a parasite infection in a subject in need thereof, comprisingadministering parasite-responsive CD4 T cells isolated from a compatiblehost (preferably of the same species as the subject), wherein the hosthas been immunized with a composition of the invention: i.e. acomposition which comprises an immunologically effective amount of (i) anucleic acid comprising a sequence which encodes a parasite MIF antigenor (ii) a parasite MIF antigen. The compatible host may have beenadministered a composition of the invention in accordance with a methodof providing protective immunity as defined herein. The compatible hostmay have been administered a composition of the invention as a singledose or in multiple doses (i.e. two or more doses) as described herein.In some embodiments, the compatible host may have been immunized withthe composition and subsequently either infected with the parasite (seebelow) or immunized with another parasite antigen (to produce theparasite-responsive CD4 T cell population). In some embodiments, thecompatible host may have been cured of the parasite infection, e.g. byadministration of an agent which kills or attenuates the parasite. Forexample, a Plasmodium infection may be cured by administration of anantimalarial. Examples of such agents/antimalarials include chloroquine(CQ), doxycycline, atovaquone (plus proguanil) and mefloquine. In someembodiments, the parasite-responsive CD4 T cells have been isolated froma compatible host who has been: (i) administered a composition of theinvention, and (ii) subsequently either infected with the parasite orimmunized with another parasite antigen (to produce theparasite-responsive CD4 T cell population). Optionally (e.g. where thehost is infected with the parasite), the host may also have been curedof the parasite infection prior to isolation of the parasite-responsiveCD4 T cells. The parasite-responsive CD4 T cells isolated from said hostmay provide the subject with sterilizing immunity (i.e. completeprotective immunity), whereby the protected subject can elicit an immuneresponse which completely eliminates the parasite infection.

The parasite may be an invertebrate parasite, for example protozoan or ahelminth. In some embodiments, the parasite is a protozoan, for examplean apicomplexan parasite such as Plasmodium. In some embodiments, theparasitic protozoan belongs to a genus selected from the groupconsisting of: Plasmodium, Toxoplasma, Babesia, Eimeria, Theileria,Neospora, Sarcocystis, Leishmania, and Trypanosoma. In some embodiments,the parasite is a helminth, for example a nematode. In some embodiments,the parasitic helminth belongs to a genus selected from the groupconsisting of: Ancyclostoma, Necator, Brugia, Wuchereria, Loa,Mansonella, Trichinella, Trichuris, Ascaris, Anisakis, Dracunculus,Strongyloides, Haemonchus, Schistosoma and Fasciola.

In a further aspect, the invention provides a composition comprising animmunologically effective amount of: (i) a nucleic acid comprising asequence which encodes a parasite MIF antigen; or (ii) a parasite MIFantigen; wherein the MIF antigen is from a parasitic protozoan. Theinvention also provides a composition comprising an immunologicallyeffective amount of: (i) a nucleic acid comprising a sequence whichencodes a parasite MIF antigen; or (ii) a parasite MIF antigen; whereinthe MIF antigen is from a parasitic helminth which belongs to a genusselected from the group consisting of: Ancyclostoma, Necator, Brugia,Wuchereria, Loa, Mansonella, Trichinella, Trichuris, Ascaris, Anisakis,Dracunculus, Strongyloides, Haemonchus, Schistosoma and Fasciola.

Parasite MIF Antigens

A parasite MIF antigen for use in the present invention generates animmune response in a subject which recognises a naturally occurringparasite MIF polypeptide (e.g. a protective immune response). Theparasite MIF antigen may also be referred to as a parasite MIFpolypeptide antigen. “Parasite MIF antigen” includes immunogenicfragments of a parasite MIF polypeptide as well as a whole orfull-length parasite MIF polypeptide. For example, a parasite MIFantigen may comprise or consist of a full-length parasite MIFpolypeptide or an immunogenic fragment of a parasite MIF polypeptide.The parasite MIF polypeptide may be a naturally occurring parasite MIFpolypeptide or a variant thereof (i.e. a variant having one or moreamino acid substitutions and/or deletions). In certain embodiments, theparasite MIF antigen comprises a contiguous amino acid sequence and/oran epitope which is found in a naturally occurring parasite MIFpolypeptide.

“Naturally occurring parasite MIF polypeptide”, as used herein, refersto a MIF polypeptide which is expressed in nature by a parasite.Typically, a naturally occurring parasite MIF polypeptide is from about110 to about 120 amino acids in length. In its mature, processed form ithas an N-terminal proline residue (formed after cleavage of methionineduring initial protein processing). A naturally occurring parasite MIFpolypeptide may have at least one biological activity selected fromtautomerase enzymatic activity and MIF-R (e.g. human CD74) bindingactivity. Naturally occurring MIF polypeptides are members of a uniquestructural superfamily characterized by forming a trimer of identicalsubunits. In naturally occurring MIF, each monomer may contain twoantiparallel alpha-helices that pack against a four-stranded beta-sheet,with each monomer having two additional beta-strands that interact withthe beta-sheets of adjacent subunits to form the interface betweenmonomers. The three beta-sheets may be arranged to form a barrelcontaining a solvent-accessible channel that runs through the centre ofthe protein along a molecular three-fold axis.

Examples of naturally occurring MIF polypeptides include:

Plasmodium falciparum MIF (UniProt Accession code Q8I5C5); SEQ ID NO: 1:PCCEVITNVNLPDDNVQSTLSQIENAISDVMGKPLGYIMSNYDYQKNLRFGGSNEAYCFVRITSIGGINRSNNSALADQITKLLVSNLNVKSRRIYVEFRDCSAQNFAFSGSLFG Plasmodium berghei MIF(UniProt Accession code Q4YQW0); SEQ ID NO: 2:PCCELITNISIPDDKAQNTLSEIEDAISNILGKPVAYIMSNYDYQKNLRFSGSNEGYCFVRLTSIGGINRSNNSLLADKITKILSNHLSVKPRRVYIEFRDCSAQNFAFSGSLFG Plasmodiumyoelii MIF(UniProt Accession code Q1HEA2); SEQ ID NO: 3:PCCELITNISIPDDKAQNALSEIEDAISNVLGKPVAYIMSNYDYQKNLRFSGSNEGYCFVRLTSIGGINRSNNSSLADKITKILSNHLGVKPRRVYIEFRDCSAQNFAFSGSLFG Plasmodium chabaudi MIF(UniProt Accession code Q4Y5M8); SEQ ID NO: 4:PCCELITNISIPDDKAQAALSEIEDAISNVLGKPTAYIMSNYDYQKNLRFAGSNEGYCFVRLTSLGGINRSNNSSLADKITKHLANHLGVKPRRVYIEFRDCSAQNFAFSGSLFG Plasmodium vivax MIF(UniProt Accession code A5K093); SEQ ID NO: 5:PCCQVSTNINASDDDAKKALSQIENAISQVLGKPLGYIMSNLDYQKHMRFGGSHDGFCFVRVTSLGGINKSNNSSLADKITKILASTLNVKSERVFIEFKDCSAQNFAFNGSLFG Plasmodium knowlesi MIF(UniProt Accession code B3LCT3); SEQ ID NO: 6:PCCQVSTNINVSDDDAKKALMQIENAISQVMNKPMGYIMSNLDYQKHMRFGGSHDGFCFVRVTSISGISRSNNTALADKITKILASTIKVKSDRVFIEFKDCSAQNFAFNGSLFG Toxoplasma gondii MIF(UniProt Accession code A1XDS9); SEQ ID NO: 7:PKCMIFCPVAATPAQQDALLKDAEKAVADALGKPLSYVMVGYSQTGQMRFGGSSDPCAFIRVASIGGITSSTNCKIAAALSAACERHLGVPKNRIYTTFTNKSPSEWAMGDRTFG Leishmania major MIF₁(UniProt Accession code Q4Q413); SEQ ID NO: 8:PVIQTFVSTPLEHHKRENLAQVYRAVTRDVLGKPEDLVMMTFHDSTPMHFFGSTDPVACVRVEALGGYGPSEPEKVTSIVTAAITK ECGIVADRIFVLYFSPLHCGWNGTNFLeishmania major MIF₂ (UniProt Accession code Q4Q412); SEQ ID NO: 9:PFLQTIVSVSLDDQKRANLSAAYGMICREELGKPEDFVMTAFSDKTPISFQGSTAPAAYVRVESWGEYAPSKPKMMTPRIAAAITK ECGIPAERIYVFYYSTKHCGWNGTNFGiardia intestinalis MIF (UniProt Accession code A8BFP4); SEQ ID NO: 10:PCAIVTTNADFTKDQADAFCLDMGQVLAKETGKPVSYCMAGVRKADMSFGTSTDLCCFVDFYCIGVISQAKNPSISAAITGCLTQH FKVKPERVYISFNEAKGHNWGFNGSTFBrugia malayi MIF (UniProt Accession code A8PJU3); SEQ ID NO: 11:PYFTIDTNIPQNSISSAFLKKASNVVAKALGKPESYVSIHVNGGQAMVFGGSEDPCAVCVLKSIGCVGPKVNNSHAEKLYKLLADE LKIPKNRCYIEFVDIEASSMAFNGSTFGWuchereria bancrofti MIF (UniProt Accession code 044786); SEQ ID NO: 12:PYFTIDTNKPQCSISSAFLKKAPNVVPKALGKPESYVSIHVNGGQPMVFGGSEDPCPVCVLKSIGCVGPKVNNSHAEKLYKLLADE LKIPKNRCYIESVDIEASSMAFNGSTFGAncylostoma duodenale MIF (UniProt Accession code I3RWR9); SEQ ID NO: 13: PMVRVATNLPDKDVPANFEERLTDILAESMNKPRNRIAIEVMAGQRITHGASRNPVAVIKVESIGALSADDNIRHTQKITQFCQDTLKLPKDKVIITYFDLQPIHVGFNGTTVAAATM Ancylostoma ceylanicum MIF₁(UniProt Accession code A4GRE3); SEQ ID NO: 14:PMVRVATNLPDKDVPANFEERLTDLLAESMNKPRNRIAIEVLAGQRITHGASRNPVAVIKVESIGALSADDNIRHTQKITQFCQDTLKLPKDKVIITYFDLQPIHVGFNGTTVAAATM Ancylostoma ceylanicum MIF₂(UniProt Accession code B6RTC1); SEQ ID NO: 15:PVFQLHTNVSQDKVTPDLLKQISALVARILHKPESYVAVHVVPDQKMTFAGTDGPCGIGILKSIGGVGGSQNNSHAKALFALIKDH LGIEGSRMYIEFVDIGASDIAHNGRTFATrichinella spiralis MIF (UniProt Accession code E5SFT7); SEQ ID NO: 16:PIFTLNTNIKATDVPSDFLSSTSALVGNILSKPGSYVAVHINTDQQLSFGGSTNPAAFGTLMSIGGIEPSRNRCHSAKLFDHLNKK LGIPKNRMYIHFVNLNGDDVGWNGTTFTrichuris trichiura MIF  (UniProt Accession code P81748); SEQ ID NO: 17:PIFTFSTNVPSENISVDFLKSTSKLIAGMLGKPESYVAVHINGGQKITFGGTDAPAGFGQLLSLGGVGGEKNRSHSAKLFKHLTDG LGIPGNRMYINFVDMRGSDVGYNGSTFOnchocerca volvulus MIF (UniProt Accession code Q963F7); SEQ ID NO: 18:PAFTINTNIPQSNVSDAFLKKASSTVAKALGKPESYVAIHVNGGQAMVFGGSTDPCAVCVLKSIGCVGPNVNNSHSEKLFKLLADE LKIPKNRCYIEFVNIDASTMAFNGSTFGThe UniProt Accession codes referred to above refer to MIF polypeptidesequences which include an N-terminal methionine that is not present inthe mature MIF polypeptide. SEQ ID NOs: 1-18 show the mature MIFpolypeptide sequence, beginning with an N-terminal proline.

A parasite MIF antigen may comprise a parasite MIF polypeptide which isa variant of a naturally occurring parasite MIF polypeptide. The variantmay comprise an amino acid sequence which is at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98% orat least 99% identical to a full-length naturally occurring parasite MIFpolypeptide, for example, to a polypeptide according to SEQ ID NO: 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.Alternatively, or in addition, the parasite MIF antigen may comprise animmunogenic fragment (i.e. an epitope-containing fragment) of a parasiteMIF polypeptide which may comprise a contiguous amino acid sequence ofat least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19 amino acids which is identical to a contiguous amino acidsequence of a naturally occurring parasite MIF polypeptide, for example,a polypeptide according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17 or 18.

In some embodiments, the parasite MIF antigen comprises an amino acidsequence which is at least 70% identical (e.g. at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98% or at least99% identical) to SEQ ID NO:1 and/or comprises a contiguous amino acidsequence of at least 8 amino acids (e.g. at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19 amino acids) which isidentical to a contiguous amino acid sequence of SEQ ID NO:1. In someembodiments, the parasite MIF antigen comprises an amino acid sequencewhich is at least 70% identical (e.g. at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98% or at least 99%identical) to SEQ ID NO:2 and/or comprises a contiguous amino acidsequence of at least 8 amino acids (e.g. at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19 amino acids) which isidentical to a contiguous amino acid sequence of SEQ ID NO:2.

Where the parasite MIF antigen is a variant of a naturally occurringparasite MIF polypeptide, the parasite MIF antigen may have one or moreamino acid (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 etc.) substitutions and/or deletions relative to thenaturally occurring parasite MIF polypeptide. The variant may have amaximum of 5, 10, 15 or 20 substitutions and/or deletions relative tothe naturally occurring parasite MIF polypeptide. The one or moresubstitutions may be conservative amino acid replacements i.e.replacements of one amino acid with another which has a related sidechain. Genetically-encoded amino acids are generally divided into fourfamilies: (1) acidic i.e. aspartate, glutamate; (2) basic i.e. lysine,arginine, histidine; (3) non-polar i.e. alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan; and (4)uncharged polar i.e. glycine, asparagine, glutamine, cystine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In general,substitution of single amino acids within these families does not have amajor effect on the biological activity. The variant may also includeone or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 etc.) amino acid insertions (e.g. each of 1, 2, 3, 4 or 5amino acids) relative to the naturally occurring parasite MIFpolypeptide. The variant may have a maximum of 5, 10, 15 or 20insertions relative to the naturally occurring parasite MIF polypeptide.

The variant may be encoded by a nucleic acid sequence which canhybridize under stringent conditions to a nucleic acid sequence thatencodes a naturally occurring parasite MIF polypeptide. Hybridizationreactions can be performed under conditions of different “stringency”.Conditions that increase stringency of a hybridization reaction arewidely known and published in the art (e.g. page 7.52 of reference 9).Examples of relevant conditions include (in order of increasingstringency): incubation temperatures of 25° C., 37° C., 50° C., 55° C.and 68° C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, 0.1×SSC(where SSC is 0.15 M NaCl and 15 mM citrate buffer) and theirequivalents using other buffer systems; formamide concentrations of 0%,25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, ormore washing steps; wash incubation times of 1, 2, or 15 minutes; andwash solutions of 6×SSC, 1×SSC, 0.1×SSC, or de-ionized water.Hybridization techniques and their optimization are well known in theart [e.g. see references 9-12, etc.]. Preferably, a nucleic acidsequence encoding a variant hybridizes under high stringency conditions(e.g. 68° C. and 0.1×SSC) to a nucleic acid sequence that encodes anaturally occurring parasite MIF polypeptide.

In certain embodiments, the parasite MIF antigen is at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids in length.

As used herein, the term “antigen” refers to a molecule containing oneor more epitopes (e.g., linear, conformational or both) that willstimulate a host's immune system to make a humoral and/or cellularantigen-specific immunological response (i.e. an immune response whichspecifically recognises a naturally occurring parasite MIF polypeptide).An “epitope” is that portion of an antigen that determines itsimmunological specificity.

The antigen, or a nucleic acid encoding the antigen, may be isolated orpurified from a natural source (i.e. a parasite of interest), but willusually be produced by recombinant or synthetic techniques, all of whichwill be familiar to those skilled in the art.

A parasite MIF antigen may comprise at least one T-cell or B-cellepitope of the naturally occurring parasite MIF polypeptide. T- andB-cell epitopes can be identified empirically (e.g. using PEPSCAN[13,14] or similar methods), or they can be predicted (e.g. using theJameson-Wolf antigenic index [15], matrix-based approaches [16],TEPITOPE [17], neural networks [18], OptiMer & EpiMer [19, 20], ADEPT[21], Tsites [22], hydrophilicity [23], antigenic index [24] or themethods disclosed in reference 25 etc.).

In certain embodiments, the parasite MIF antigen is capable of elicitinga T cell response in the subject, for example a helper (CD4) T cellresponse.

Multiple Parasite MIF Antigens

A composition of the invention may use, or may target, a single parasiteMIF antigen, or may use or target two or more different parasite MIFantigens. Thus, in some embodiments, a composition as defined herein maycomprise or encode a single parasite MIF antigen or two or moredifferent parasite MIF antigens, and/or may comprise antibodies whichspecifically bind to a single parasite MIF antigen or to two or moredifferent parasite MIF antigens.

In some embodiments, the composition may comprise a nucleic acid whichencodes two or more parasite MIF antigens. In some embodiments, thecomposition may comprise two or more nucleic acids which each encode aparasite MIF antigen. In some embodiments, the composition may comprisetwo or more parasite MIF antigens. In some embodiments, the compositionmay comprise two or more antibodies which each respectively bind to twoor more parasite MIF antigens.

In some embodiments, a composition of the invention may comprise acombination of. (i) a nucleic acid comprising a sequence which encodes aparasite MIF antigen; (ii) a parasite MIF antigen; (iii) an antibodywhich specifically binds to a parasite MIF antigen. For example, thecomposition may comprise a combination of (i) and (ii), (i) and (iii),(ii) and (iii), or (i), (ii) and (iii). In each of (i), (ii) and (iii),the parasite MIF antigen may be a different parasite MIF antigen.

The different parasite MIF antigens may be derived from differentparasite species, may be different variants of a parasite MIF antigen,and/or may comprise different parasite MIF epitopes. In someembodiments, a composition may comprise or encode two, three, four, ormore, parasite MIF antigens that may contain a range of epitopes. Insome embodiments, a composition may comprise two, three, four, or moreantibodies which each specifically bind to a different parasite MIFantigen epitope.

Additional Parasite Antigens

As discussed above, the immune response to the parasite MIF antigen mayenhance the development of a protective immune response (e.g. a CD4memory T cell response) to one or more additional parasite antigens.Thus, parasite MIFs may be used either as stand-alone antigens or incombination with additional parasite antigens in order to promotelong-lasting memory T cell responses and protective immunity againstparasite infection.

Thus, a composition as defined herein (i.e. a composition comprising (i)a nucleic acid comprising a sequence which encodes a parasite MIFantigen, (ii) a parasite MIF antigen, or (iii) an antibody whichspecifically binds to a parasite MIF antigen) may further comprise orencode one or more additional parasite antigens (i.e. parasite antigenswhich are not parasite MIF antigens, or “non-MIF parasite antigens”). Insome embodiments, the composition may comprise a nucleic acid sequencewhich encodes an additional parasite antigen. For example, thecomposition may comprise a nucleic acid comprising both a sequence whichencodes a parasite MIF antigen and a sequence which encodes anadditional parasite antigen (i.e. the parasite MIF antigen and theadditional parasite antigen may be encoded by the same nucleic acidmolecule). Alternatively, or in addition, the composition may comprise afurther nucleic acid comprising a sequence which encodes an additionalparasite antigen (i.e. the parasite MIF antigen and the additionalparasite antigen may be encoded by separate nucleic acid molecules).

In some embodiments, the composition may comprise both a parasite MIFantigen and an additional parasite antigen.

Alternatively, or in addition, a composition as defined herein (i.e. acomposition comprising (i) a nucleic acid comprising a sequence whichencodes a parasite MIF antigen, (ii) a parasite MIF antigen, or (iii) anantibody which specifically binds to a parasite MIF antigen) may beadministered to a subject in combination with a further compositionwhich comprises or encodes one or more additional parasite antigens. Thefurther composition may be a parasite vaccine composition. For example,the one or more additional parasite antigens may be formulated as aparasite vaccine composition. In some embodiments, a composition asdefined herein may be administered to a subject in combination with afurther composition which comprises a nucleic acid comprising a sequencewhich encodes an additional parasite antigen. In some embodiments, acomposition as defined herein may be administered to a subject incombination with a further composition which comprises an additionalparasite antigen.

Accordingly, in some embodiments, a method for providing protectiveimmunity against a parasite infection in a subject in need thereofaccording to the present invention may further comprise administering tothe subject a further composition which comprises a nucleic acidcomprising a sequence which encodes an additional parasite antigen. Insome embodiments, a method for providing protective immunity against aparasite infection in a subject in need thereof according to the presentinvention may further comprise administering to the subject a furthercomposition which comprises an additional parasite antigen.

Thus, in a method for providing protective immunity against a parasiteinfection in a subject in need thereof according to the presentinvention, the subject may be administered: (1) a composition as definedherein which comprises a nucleic acid comprising a sequence whichencodes a parasite MIF antigen and a further composition which comprisesa nucleic acid comprising a sequence which encodes an additionalparasite antigen; (2) a composition as defined herein which comprises anucleic acid comprising a sequence which encodes a parasite MIF antigenand a further composition which comprises an additional parasiteantigen; (3) a composition as defined herein which comprises a parasiteMIF antigen and a further composition which comprises a nucleic acidcomprising a sequence which encodes an additional parasite antigen; (4)a composition as defined herein which comprises a parasite MIF antigenand a further composition which comprises an additional parasiteantigen; (5) a composition as defined herein which comprises an antibodywhich specifically binds to a parasite MIF antigen and a furthercomposition which comprises a nucleic acid comprising a sequence whichencodes an additional parasite antigen; or (6) a composition as definedherein which comprises an antibody which specifically binds to aparasite MIF antigen and a further composition which comprises anadditional parasite antigen.

The composition as defined herein (i.e. a composition comprising (i) anucleic acid comprising a sequence which encodes a parasite MIF antigen,(ii) a parasite MIF antigen, or (iii) an antibody which specificallybinds to a parasite MIF antigen) and the composition comprising orencoding the additional parasite antigen may be provided as separatecomponents and/or administered separately. Alternatively, thecomposition as defined herein (i.e. a composition comprising (i) anucleic acid comprising a sequence which encodes a parasite MIF antigen,(ii) a parasite MIF antigen, or (iii) an antibody which specificallybinds to a parasite MIF antigen) and the composition comprising orencoding the additional parasite antigen may be mixed prior toadministration.

Administration of (A) the composition as defined herein (i.e. acomposition comprising (i) a nucleic acid comprising a sequence whichencodes a parasite MIF antigen, (ii) a parasite MIF antigen, or (iii) anantibody which specifically binds to a parasite MIF antigen) and (B) thecomposition comprising or encoding the additional parasite antigen, inany combination as described herein, may be contemporaneous. Forexample, compositions (A) and (B) may be administered simultaneously,separately or sequentially. Compositions (A) and (B) may be administeredwithin 12 months of each other, within six months of each other, orwithin one month or less of each other (e.g. within 10 days).Compositions (A) and (B) may be administered within 7 days, within 3days, within 2 days, or within 24 hours of each other. Simultaneousadministration may involve administering compositions (A) and (B) at thesame time. Simultaneous administration may include administration of (A)and (B) to a patient within 12 hours of each other, within 6 hours,within 3 hours, within 2 hours or within 1 hour of each other, typicallywithin the same visit to a clinical centre. Composition (A) may beadministered before (B).

In certain embodiments, the additional parasite antigen may be derivedfrom the same parasite species as the parasite MIF antigen. In otherembodiments, the additional parasite antigen may be from a differentspecies compared to the parasite MIF antigen.

The one or more additional parasite antigens may, for example, bePlasmodium antigens, or derived from Plasmodium antigens, such ascircumsporozoite (CS) protein (optionally fused to a hepatitis B surfaceantigen (HBsAg)); merozoite surface protein (MSP), for example MSP-1;reticulocyte-binding protein homologue 5 (RH5), for example, PfRH5;apical membrane antigen 1 (AMA1); thrombospondin-related adhesionprotein (TRAP); ME-TRAP (multiple epitope string withthrombospondin-related adhesion protein: a pre-erythrocytic fusionantigen consisting of 17 B cell, CD4+ and CD8+ T cell epitopes from sixP. falciparum antigens fused to the T9/96 allele of TRAP); liver-stageantigen 1 (LSA-1); liver stage antigen-3 (LSA-3); exported protein 1(Exp-1); antigen encoded by polyepitope DNA EP1300 with linker sequencesfrom four pre-erythrocytic antigens, CS, TRAP, LSA-1 and Exp-1;polyprotein comprising LSA-3, sporozoite threonine and asparagine richprotein (STARP), Exp1, Pfs16, TRAP, and LSA-1; a falciparum merozoiteprotein (FMP) such as FMP010 or FMP001; merozoite surface protein-3(MSP-3); erythrocyte binding antigen-175 (EBA175); EBA175 RII; serinerepeat antigen (SERA5); SE36, a recombinant protein corresponding to afragment of the SERA5 antigen; glutamate-rich protein (GLURP);ring-infected erythrocyte surface antigen (RESA); antigenic fragments ofany one of the foregoing; or a combination thereof. In some embodiments,the one or more additional antigens may be in the form ofnon-replicating sporozoites, such as PfSPZ (which is composed ofattenuated, aseptic, purified, cryopreserved Plasmodium falciparumsporozoites) (e.g. Seder et al. Science 341, 1359 (2013)).

In some embodiments, an additional parasite antigen for use with theinvention is a CS protein, which includes P. falciparum antigens basedon the circumsporozoite (CS) protein. This can take the form of arecombinant protein that fuses a part of the CS protein with HBsAg,known as ‘RTS’, or TRAP. Suitable P. falciparum antigens for makingHBsAg hybrids may be based on a subunit of the circumsporozoite surfaceantigen (“CSP”) e.g. they may include between 3 and 20 repeats of itsNANP (SEQ ID NO: 19) motif, and/or they may include the C-terminalregion of CSP (but typically not including the final 12 amino acids fromthe C-terminus). RTS is a hybrid protein comprising substantially allthe C-terminal portion of CS from the NF54 or 7G8 isolate of P.falciparum (amino acids 210 to 398, which includes 19 NANP (SEQ ID NO:19) repeats and the T cell epitope region at amino acids 367 to 390),fused to the N-terminus of HBsAg by four amino acids of the preS2portion of HBsAg [26]. The sequence of RTS can thus contain: (i) aN-terminus methionine residue; (ii) Met-Ala-Pro; (iii) 189 amino acidscorresponding either to amino acids 210-398 of CS protein from P.falciparum 7G8 or to amino acids 207-395 of CS protein from P.falciparum NF54; (iv) Arg or Gly; (v) Pro-Val-Thr-Asn from hepatitis BPre-S2 protein; and (vi) HBsAg. When expressed in yeast (particularly inS. cerevisiae) RTS is produced as a lipoprotein particle (including inparticular phospholipids), and when it is co-expressed with the Santigen from HBV it produces a mixed particle known as RTS,S. A RTS:Sratio of about 1:4 is useful. TRAP antigens are described in reference27. In some embodiments, an additional parasite antigen may take theform of RTS,S.

The one or more additional parasite antigens may be derived from anyspecies of Plasmodium, including any of the Plasmodium species listedbelow. In some embodiments, the additional parasite antigen is aPlasmodium falciparum or Plasmodium vivax antigen. In some embodiments,the additional parasite antigen is CS protein (optionally fused toHBsAg); MSP-1; PfRH5; AMA1; and antigenic fragments thereof. Forexample, the parasite MIF antigen may be a Plasmodium falciparum: MIFantigen and the additional parasite antigen may be a Plasmodiumfalciparum circumsporozoite (CS) protein fused to a hepatitis B surfaceantigen (HBsAg).

In further embodiments, the one or more additional parasite antigens maybe Leishmania antigens, or derived from Leishmania antigens, such asthiol-specific antioxidant (TSA); stress-inducible protein 1 (LmSTI1);Leishmania elongation initiation factor (LeIF); recombinant surfaceantigen gp63; lipophosphoglycan; a 46 kD promastigote antigen derivedfrom L. amazonensis; Leishmania-activated C kinase (LACK); parasitesurface antigen (PSA); and parasite surface antigen-2 (PSA-2);Schistosoma antigens such as 63 kD parasite myosin; 97 kD paramyosin; 28kD triose phosphate isomerase (TPI); 23 kD integral membrane protein(Sm23); 26 and 28 kD GST; 28 kD S. haematobium GST (Sh28GST);Tetraspanin-2 (SmTSP-2) and fatty acid binding protein (FABP); may beAncyclostoma antigens, or derived from Ancylostoma antigens, such asAncylostoma-secreted protein (ASP); may be Necator antigens, or derivedfrom Necator antigens, such as Na-ASP-2, a 21 kDa protein from Necatoramericanus; antigenic fragments of any one of the foregoing, or acombination thereof.

A composition of the invention may also be used in a method of enhancingan immune response to another (i.e. non-MIF) parasite antigen (e.g.another Plasmodium antigen, as described herein). Thus, provided hereinis a method of enhancing an immune response to a parasite antigencomprising administering a composition of the invention (i.e. acomposition comprising (i) a nucleic acid comprising a sequence whichencodes a parasite MIF antigen, (ii) a parasite MIF antigen, and/or(iii) an antibody which specifically binds to a parasite MIF antigen) toa subject. The parasite antigen against which the immune response isenhanced may include one or more additional parasite antigens, asdefined herein. The method may further comprise administering thenon-MIF parasite antigen to the subject. The MIF and non-MIF antigenscan be administered simultaneously, separately, or sequentially.

The immune response may be enhanced relative to the immune response in asubject treated with only the non-MIF parasite antigen. The enhancedimmune response may comprise an enhanced protective immune responseagainst the parasite infection. Protective immune responses are definedherein. For example, the protective immune response may be characterizedby protective immunological memory (e.g. a protective memory T cellresponse) against the parasite. Protective immunity may be sterilizingimmunity.

Polypeptides

In some embodiments, a polypeptide according to the present invention isin a non-naturally occurring form (e.g. a recombinant or modified form).

For example, polypeptides (e.g. antigens) disclosed herein can beprepared by chemical synthesis (in whole or in part), by digestinglonger polypeptides using proteases, by translation from RNA, bypurification from cell culture (e.g. from recombinant expression), fromthe organism itself, etc. An exemplary method for production of peptides<40 amino acids long involves in vitro chemical synthesis [28,29].Solid-phase peptide synthesis techniques, such as methods based on tBocor Fmoc [30] chemistry, are known in the art. Enzymatic synthesis [31]may also be used in part or in full. As an alternative to chemicalsynthesis, biological synthesis may be used e.g. the polypeptides may beproduced by translation. This may be carried out in vitro or in vivo.Biological methods are in general restricted to the production ofpolypeptides based on L-amino acids, but manipulation of translationmachinery (e.g. of aminoacyl tRNA molecules) can be used to allow theintroduction of D-amino acids (or of other non-natural amino acids, suchas iodotyrosine or methylphenylalanine, azidohomoalanine, etc.) [32].Where D-amino acids are included, however, it is preferred to usechemical synthesis. Polypeptides of the disclosure may have covalentmodifications at the C-terminus and/or N-terminus. They can also takevarious forms (e.g. native, fusions, glycosylated, non-glycosylated,lipidated, non-lipidated, phosphorylated, non-phosphorylated,myristoylated, non-myristoylated, monomeric, multimeric, particulate,denatured, etc.). The polypeptides can be naturally or non-naturallyglycosylated (i.e. the polypeptide may have a glycosylation pattern thatdiffers from the glycosylation pattern found in the correspondingnaturally occurring polypeptide).

Non-naturally occurring forms of polypeptides according to the inventionmay comprise one or more heterologous amino acid sequences (e.g. anotherantigen sequence or a detectable tag) in addition to a parasite MIFantigen sequence. For example, a polypeptide of the invention may be afusion protein. Alternatively, or in addition, the amino acid sequenceor chemical structure of the polypeptide may be modified (e.g. with oneor more non-natural amino acids, by covalent modification, and/or or byhaving a different glycosylation pattern, for example, by the removal oraddition of one or more glycosyl groups) compared to anaturally-occurring polypeptide sequence.

Polypeptides (e.g. antigens) disclosed herein are preferably provided inpurified or substantially purified form i.e. substantially free fromother polypeptides (e.g. free from naturally-occurring polypeptides),particularly from other parasite or host cell polypeptides; for example,at least about 50% pure (by weight), at least about 60% pure (byweight), at least about 70% pure (by weight), at least about 80% pure(by weight), or at least about 90% pure, etc. Alternatively, less thanabout 50%, less than about 40%, less than about 30%, less than about20%, less than about 10%, or less than about 5% of a composition is madeup of other expressed polypeptides.

Nucleic Acids

The invention also relates to nucleic acid comprising a sequence whichencodes a parasite MIF antigen, as disclosed herein. Nucleic acidaccording to the invention can take various forms (e.g. single-stranded,double-stranded, vectors etc.). Nucleic acids of the invention may becircular or branched, but will generally be linear.

The nucleic acids used in the invention are preferably provided inpurified or substantially purified form i.e. substantially free fromother nucleic acids (e.g. free from naturally-occurring nucleic acids),particularly from other parasite or host cell nucleic acids, generallybeing at least about 50% pure (by weight), and usually at least about90% pure.

Nucleic acids may be prepared in many ways e.g. by chemical synthesis(e.g. phosphoramidite synthesis of DNA) in whole or in part, bydigesting longer nucleic acids using nucleases (e.g. restrictionenzymes), by joining shorter nucleic acids or nucleotides (e.g. usingligases or polymerases), from genomic or cDNA libraries, etc.

The term “nucleic acid” in general means a polymeric form of nucleotidesof any length, which contain deoxyribonucleotides, ribonucleotides,and/or their analogs. It includes DNA, RNA, DNA/RNA hybrids. It alsoincludes DNA or RNA analogs, such as those containing modified backbones(e.g. peptide nucleic acids (PNAs) or phosphorothioates) or modifiedbases. Thus the nucleic acid of the disclosure includes mRNA, DNA, cDNA,recombinant nucleic acids, branched nucleic acids, plasmids, vectors,etc. Where the nucleic acid takes the form of RNA, it may or may nothave a 5′ cap.

The nucleic acids of the invention comprise a sequence which encodes atleast one parasite MIF antigen. Typically, the nucleic acids of theinvention will be in recombinant form, i.e. a form which does not occurin nature. For example, the nucleic acid may comprise one or moreheterologous nucleic acid sequences (e.g. a sequence encoding anotherantigen and/or a control sequence such as a promoter or an internalribosome entry site) in addition to the sequence encoding at least oneparasite MIF antigen. The nucleic acid may be part of a vector i.e. partof a nucleic acid construct designed for transduction/transfection ofone or more cell types. Vectors may be, for example, “expressionvectors” which are designed for expression of a nucleotide sequence in ahost cell, or “viral vectors” which are designed to result in theproduction of a recombinant virus or virus-like particle.

Alternatively, or in addition, the sequence or chemical structure of thenucleic acid may be modified compared to a naturally-occurring sequencewhich encodes a parasite MIF antigen. The sequence of the nucleic acidmolecule may be modified, e.g. to increase the efficacy of expression orreplication of the nucleic acid, or to provide additional stability orresistance to degradation. For example, the sequence of the nucleic acidmolecule may be codon optimized for expression in a desired host, suchas a mammalian (e.g. human) cell. Such modification with respect tocodon usage may increase translation efficacy and half-life of thenucleic acid. A poly A tail (e.g., of about 30 adenosine residues ormore) may be attached to the 3′ end of the RNA to increase itshalf-life. The 5′ end of the RNA may be capped with a modifiedribonucleotide with the structure m7G (5′) ppp (5′) N (cap 0 structure)or a derivative thereof, which can be incorporated during RNA synthesisor can be enzymatically engineered after RNA transcription (e.g., byusing Vaccinia Virus Capping Enzyme (VCE) consisting of mRNAtriphosphatase, guanylyl-transferase and guanine-7-methytransferase,which catalyzes the construction of N7-monomethylated cap 0 structures).Cap 0 structure plays an important role in maintaining the stability andtranslational efficacy of the RNA molecule. The 5′ cap of the RNAmolecule may be further modified by a 2′-O-Methyltransferase whichresults in the generation of a cap 1 structure (m7Gppp [m2′-O] N), whichmay further increases translation efficacy.

The nucleic acids may comprise one or more nucleotide analogs ormodified nucleotides. As used herein, “nucleotide analog” or “modifiednucleotide” refers to a nucleotide that contains one or more chemicalmodifications (e.g., substitutions) in or on the nitrogenous base of thenucleoside (e.g., cytosine (C), thymine (T) or uracil (U)), adenine (A)or guanine (G)). A nucleotide analog can contain further chemicalmodifications in or on the sugar moiety of the nucleoside (e.g., ribose,deoxyribose, modified ribose, modified deoxyribose, six-membered sugaranalog, or open-chain sugar analog), or the phosphate. The preparationof nucleotides and modified nucleotides and nucleosides are well-knownin the art, e.g. from U.S. Pat. Nos. 4,373,071, 4,458,066, 4,500,707,4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530,5,700,642, and many modified nucleosides and modified nucleotides arecommercially available.

Modified nucleobases which can be incorporated into modified nucleosidesand nucleotides and be present in the RNA molecules include: m5C(5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U(2-thiouridine), Um (2′-O-methyluridine), m1A (1-methyladenosine); m2A(2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A(2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A(2-methylthio-N6isopentenyladenosine); io6A(N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine);ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine);hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m1I (1-methylinosine);m′Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm(2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine);f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm(N4acetyl2TOmethylcytidine); k2C (lysidine); mlG (1-methylguanosine);m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm(2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm(N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-O-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q(queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi(7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine);m5Um (5,2′-O-dimethyluridine); s4U (4-thiouridine); m5s2U(5-methyl-2-thiouridine); s2Um (2-thio-2′-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U(5-carboxymethylaminomethyluridine); cnmm5Um(5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tm (2′-O-methylinosine); m4C(N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U(5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am(N6,N6,O-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine); m2′2′7G(N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D(5-methyldihydrouridine); f5Cm (5-formyl-2′-O-methylcytidine); m1Gm(1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyl adenosine)irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14(4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine),hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof,dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,5-(C₁-C₆)-alkyluracil, 5-methyluracil, 5-(C₂-C₆)-alkenyluracil,5-(C₂-C₆)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil,5-fluorouracil, 5-bromouracil, 5-hydroxycytosine,5-(C₁-C₆)-alkylcytosine, 5-methylcytosine, 5-(C₂-C₆)-alkenylcytosine,5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, N²-dimethylguanine, 7-deazaguanine, 8-azaguanine,7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine,7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine,8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine,2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine,7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen(abasic residue), m5C, m5U, m6A, s2U, W, or 2′-O-methyl-U. Many of thesemodified nucleobases and their corresponding ribonucleosides areavailable from commercial suppliers. See, e.g., WO 2011/005799.

Nucleic Acid-Based Vaccines

A composition as disclosed herein comprising a nucleic acid sequencewhich encodes a parasite MIF antigen may be a nucleic acid-basedvaccine. A further composition comprising a nucleic acid sequence whichencodes one or more additional parasite antigens may also be provided asa nucleic acid-based vaccine.

The nucleic acid may, for example, be RNA (i.e. an RNA-based vaccine) orDNA (i.e. a DNA-based vaccine, such as a plasmid DNA vaccine). Incertain embodiments, the nucleic acid-based vaccine is an RNA-basedvaccine. In certain embodiments, the RNA-based vaccine comprises aself-replicating RNA molecule. The self-replicating RNA molecule may bean alphavirus-derived RNA replicon.

Self-replicating RNA molecules are well known in the art and can beproduced by using replication elements derived from, e.g., alphaviruses,and substituting the structural viral proteins with a nucleotidesequence encoding a protein of interest. A self-replicating RNA moleculeis typically a +−strand molecule which can be directly translated afterdelivery to a cell, and this translation provides a RNA-dependent RNApolymerase which then produces both antisense and sense transcripts fromthe delivered RNA. Thus the delivered RNA leads to the production ofmultiple daughter RNAs. These daughter RNAs, as well as collinearsubgenomic transcripts, may be translated themselves to provide in situexpression of an encoded antigen (i.e. a parasite MIF antigen), or maybe transcribed to provide further transcripts with the same sense as thedelivered RNA which are translated to provide in situ expression of theantigen. The overall result of this sequence of transcriptions is a hugeamplification in the number of the introduced replicon RNAs and so theencoded antigen becomes a major polypeptide product of the cells.

One suitable system for achieving self-replication in this manner is touse an alphavirus-based replicon. These replicons are +−stranded RNAswhich lead to translation of a replicase (or replicase-transcriptase)after delivery to a cell. The replicase is translated as a polyproteinwhich auto-cleaves to provide a replication complex which createsgenomic −−strand copies of the +−strand delivered RNA. These −−strandtranscripts can themselves be transcribed to give further copies of the+−stranded parent RNA and also to give a subgenomic transcript whichencodes the antigen. Translation of the subgenomic transcript thus leadsto in situ expression of the antigen by the infected cell. Suitablealphavirus replicons can use a replicase from a Sindbis virus, a Semlikiforest virus, an eastern equine encephalitis virus, a Venezuelan equineencephalitis virus, etc. Mutant or wild-type virus sequences can be usede.g. the attenuated TC83 mutant of VEEV has been used in replicons [33].

In certain embodiments, the self-replicating RNA molecule describedherein encodes (i) a RNA-dependent RNA polymerase which can transcribeRNA from the self-replicating RNA molecule and (ii) a parasite MIFantigen. The polymerase can be an alphavirus replicase e.g. comprisingone or more of alphavirus proteins nsP1, nsP2, nsP3 and nsP4.

Whereas natural alphavirus genomes encode structural virion proteins inaddition to the non-structural replicase polyprotei, in certainembodiments, the self-replicating RNA molecules do not encode alphavirusstructural proteins. Thus, the self-replicating RNA can lead to theproduction of genomic RNA copies of itself in a cell, but not to theproduction of RNA-containing virions. The inability to produce thesevirions means that, unlike a wild-type alphavirus, the self-replicatingRNA molecule cannot perpetuate itself in infectious form. The alphavirusstructural proteins which are necessary for perpetuation in wild-typeviruses are absent from self-replicating RNAs of the present disclosureand their place is taken by gene(s) encoding the immunogen of interest,such that the subgenomic transcript encodes the immunogen rather thanthe structural alphavirus virion proteins.

Thus a self-replicating RNA molecule useful with the invention may havetwo open reading frames. The first (5′) open reading frame encodes areplicase; the second (3′) open reading frame encodes an antigen. Insome embodiments the RNA may have additional (e.g. downstream) openreading frames e.g. to encode further antigens or to encode accessorypolypeptides.

In certain embodiments, the self-replicating RNA molecule disclosedherein has a 5′ cap (e.g. a 7-methylguanosine). This cap can enhance invivo translation of the RNA. In some embodiments the 5′ sequence of theself-replicating RNA molecule must be selected to ensure compatibilitywith the encoded replicase.

A self-replicating RNA molecule may have a 3′ poly-A tail. It may alsoinclude a poly-A polymerase recognition sequence (e.g. AAUAAA) near its3′ end.

Self-replicating RNA molecules can have various lengths but they aretypically 5000-25000 nucleotides long. Self-replicating RNA moleculeswill typically be single-stranded. Single-stranded RNAs can generallyinitiate an adjuvant effect by binding to TLR7, TLR8, RNA helicasesand/or PKR. RNA delivered in double-stranded form (dsRNA) can bind toTLR3, and this receptor can also be triggered by dsRNA which is formedeither during replication of a single-stranded RNA or within thesecondary structure of a single-stranded RNA.

The self-replicating RNA can conveniently be prepared by in vitrotranscription (IVT). IVT can use a (cDNA) template created andpropagated in plasmid form in bacteria, or created synthetically (forexample by gene synthesis and/or polymerase chain-reaction (PCR)engineering methods). For instance, a DNA-dependent RNA polymerase (suchas the bacteriophage T7, T3 or SP6 RNA polymerases) can be used totranscribe the self-replicating RNA from a DNA template. Appropriatecapping and poly-A addition reactions can be used as required (althoughthe replicon's poly-A is usually encoded within the DNA template). TheseRNA polymerases can have stringent requirements for the transcribed 5′nucleotide(s) and in some embodiments these requirements must be matchedwith the requirements of the encoded replicase, to ensure that theIVT-transcribed RNA can function efficiently as a substrate for itsself-encoded replicase.

A self-replicating RNA can include (in addition to any 5′ cap structure)one or more nucleotides having a modified nucleobase. A RNA used withthe invention ideally includes only phosphodiester linkages betweennucleosides, but in some embodiments it can contain phosphoramidate,phosphorothioate, and/or methylphosphonate linkages.

The self-replicating RNA molecule may encode a single heterologouspolypeptide antigen (i.e. a parasite MIF antigen) or, optionally, two ormore heterologous polypeptide antigens linked together in a way thateach of the sequences retains its identity (e.g., linked in series) whenexpressed as an amino acid sequence. The heterologous polypeptidesgenerated from the self-replicating RNA may then be produced as a fusionpolypeptide or engineered in such a manner to result in separatepolypeptide or peptide sequences.

The self-replicating RNA molecules described herein may be engineered toexpress multiple nucleotide sequences, from two or more open readingframes, thereby allowing co-expression of proteins, such as one, two ormore parasite antigens (e.g. one, two or more parasite MIF antigens)together with cytokines or other immunomodulators, which can enhance thegeneration of an immune response. Such a self-replicating RNA moleculemight be particularly useful, for example, in the production of variousgene products (e.g., proteins) at the same time, for example, as abivalent or multivalent vaccine.

If desired, the self-replicating RNA molecules can be screened oranalyzed to confirm their therapeutic and prophylactic properties usingvarious in vitro or in vivo testing methods that are known to those ofskill in the art. For example, vaccines comprising self-replicating RNAmolecule can be tested for their effect on induction of proliferation oreffector function of the particular lymphocyte type of interest, e.g., Bcells, T cells, T cell lines, and T cell clones. For example, spleencells from immunized mice can be isolated and the capacity of cytotoxicT lymphocytes to lyse autologous target cells that contain aself-replicating RNA molecule that encodes a parasite MIF antigen. Inaddition, T helper cell differentiation can be analyzed by measuringproliferation or production of TH1 (IL-2 and IFN-γ) and/or TH2 (IL-4 andIL-5) cytokines by ELISA or directly in CD4+ T cells by cytoplasmiccytokine staining and flow cytometry.

Self-replicating RNA molecules that encode a parasite MIF antigen canalso be tested for ability to induce humoral immune responses, asevidenced, for example, by induction of B cell production of antibodiesspecific for a parasite MIF antigen of interest. These assays can beconducted using, for example, peripheral B lymphocytes from immunizedindividuals. Such assay methods are known to those of skill in the art.Other assays that can be used to characterize the self-replicating RNAmolecules can involve detecting expression of the encoded parasite MIFantigen by the target cells. For example, FACS can be used to detectantigen expression on the cell surface or intracellularly. Anotheradvantage of FACS selection is that one can sort for different levels ofexpression; sometimes-lower expression may be desired. Other suitablemethod for identifying cells which express a particular antigen involvepanning using monoclonal antibodies on a plate or capture using magneticbeads coated with monoclonal antibodies.

Suitable types of nucleic acid-based vaccine for use according to thepresent disclosure are described in references 34, 35 and 36.

The nucleic acid-based vaccine may comprise a viral or a non-viraldelivery system. The delivery system (also referred to herein as adelivery vehicle) may have adjuvant effects which enhance theimmunogenicity of the encoded parasite MIF antigen. For example, thenucleic acid molecule may be encapsulated in liposomes, non-toxicbiodegradable polymeric microparticles or viral replicon particles(VRPs), or complexed with particles of a cationic oil-in-water emulsion.In some embodiments, the nucleic acid-based vaccine comprises a cationicnano-emulsion (CNE) delivery system or a lipid nanoparticle (LNP)delivery system. Alternatively, the nucleic acid-based vaccine maycomprise viral replicon particles. In other embodiments, the nucleicacid-based vaccine may comprise a naked nucleic acid, such as naked RNA(e.g. mRNA), but delivery via LNPs is preferred.

In certain embodiments, the nucleic acid-based vaccine comprises acationic nano-emulsion (CNE) delivery system. CNE delivery systems andmethods for their preparation are described in reference 35. In a CNEdelivery system, the nucleic acid molecule (e.g. RNA) which encodes theantigen is complexed with a particle of a cationic oil-in-wateremulsion. Cationic oil-in-water emulsions can be used to delivernegatively charged molecules, such as an RNA molecule to cells. Theemulsion particles comprise an oil core and a cationic lipid. Thecationic lipid can interact with the negatively charged molecule therebyanchoring the molecule to the emulsion particles. Further details ofuseful CNEs can be found in references 35, 37 & 38 (the completecontents of all of which are incorporated by reference herein).

Thus, in a nucleic acid-based vaccine of the invention, an RNA moleculeencoding a parasite MIF antigen may be complexed with a particle of acationic oil-in-water emulsion. The particles typically comprise an oilcore (e.g. a plant oil or squalene) that is in liquid phase at 25° C., acationic lipid (e.g. phospholipid) and, optionally, a surfactant (e.g.sorbitan trioleate, polysorbate 80); polyethylene glycol can also beincluded. In some embodiments, the CNE comprises squalene and a cationiclipid, such as 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP). Insome preferred embodiments, the delivery system is a non-viral deliverysystem, such as CNE, and the nucleic acid-based vaccine comprises aself-replicating RNA (mRNA). This may be particularly effective ineliciting humoral and cellular immune responses. Advantages also includethe absence of a limiting anti-vector immune response and a lack of riskof genomic integration.

LNP delivery systems and non-toxic biodegradable polymericmicroparticles, and methods for their preparation are described inreferences 34 and 36. LNPs are non-virion liposome particles in which anucleic acid molecule (e.g. RNA) can be encapsulated. The particles caninclude some external RNA (e.g. on the surface of the particles), but atleast half of the RNA (and ideally all of it) is encapsulated. Liposomalparticles can, for example, be formed of a mixture of zwitterionic,cationic and anionic lipids which can be saturated or unsaturated, forexample; DSPC (zwitterionic, saturated), DlinDMA (cationic,unsaturated), and/or DMG (anionic, saturated). Preferred LNPs for usewith the invention include an amphiphilic lipid which can formliposomes, optionally in combination with at least one cationic lipid(such as DOTAP, DSDMA, DODMA, DLinDMA, DLenDMA, etc.). A mixture ofDSPC, DlinDMA, PEG-DMG and cholesterol is particularly effective. Otheruseful LNPs are disclosed in references 34 and 39-43 (the completecontents of all of which are incorporated by reference herein). In someembodiments, the LNPs are RV01 liposomes (references 34 and 36).

Antibodies

In one aspect, the invention relates to an antibody which specificallybinds to a parasite MIF antigen, as disclosed herein. Preferably, theantibody specifically binds to a naturally occurring parasite MIFantigen.

An antibody that “specifically binds” to a parasite MIF antigen is anantibody that binds this antigen with greater affinity and/or aviditythan it binds to other parasite or non-parasite antigens. For example,the antibody which specifically binds to a parasite MIF antigen may bindthe parasite MIF antigen with greater affinity and/or avidity than itbinds to HSA. Preferably, the antibody does not specifically bind tovertebrate MIF or a MIF antigen produced by the subject.

As used herein, the term “antibody” includes full-length or wholeantibodies (i.e. antibodies in their substantially intact form),antibody fragments such as F(ab′)2, F(ab) and Fab′-SH fragments, Fvfragments (non-covalent heterodimers), single-chain antibodies such assingle chain Fv molecules (scFv) or those derived from camelids andsharks (e.g. heavy chain antibodies), single-domain antibodies (dAbs),diabodies, minibodies, oligobodies, etc. The term “antibody” does notimply any particular origin, and includes antibodies obtained throughnon-conventional processes, such as phage display. All of the antibodieswill comprise the antigen binding site of a full-length or wholeantibody and thus retain the ability of bind antigen. Thus, the term“antibody” includes antigen-binding fragments of full-length or wholeantibodies. The antibody is ideally a monoclonal antibody, or,alternatively, may be polyclonal. The antibody may be chimeric,humanized (e.g. refs. 44 & 45), or fully human. In compositions of theinvention, polyclonal antibody, comprising one or more antibodies whichspecifically bind to the parasite MIF antigen, may be used. In somepreferred embodiments, the composition comprises polyclonal antibody,for example serum anti-parasite MIF antibody. The polyclonal antibodymay comprise IgG (e.g. purified serum IgG). The antibody may comprise aneutralizing antibody (i.e. an antibody which neutralizes the biologicaleffects of the parasite MIF in the subject).

The antibody is preferably provided in purified or substantiallypurified form. Typically, the antibody will be present in a compositionthat is substantially free of other polypeptides e.g. where less than90% (by weight), usually less than 60% and more usually less than 50% ofthe composition is made up of other polypeptides.

The antibodies can be of any isotype (e.g. IgA, IgG, IgM i.e. an α, γ orμ heavy chain), but will generally be IgG. Within the IgG isotype,antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass. The antibody mayhave a κ or a λ light chain.

Parasites

The present invention relates to treatment of parasitic infections usingparasite MIF antigens. The parasite (i.e. the causative agent of theparasitic infection and the parasite from which the parasite MIF antigenis derived) may be an invertebrate parasite, for example protozoan or ahelminth. The parasite may, for example, be a blood-borne parasite (i.e.a parasite having a life-cycle which involves a blood-borne stage).Parasites according to the invention necessarily express a MIF ortholog(i.e. a naturally occurring MIF polypeptide).

Parasitic protozoans include apicomplexan parasites, such as Plasmodiumspp., Toxoplasma spp., Eimeria spp., Babesia spp., Theileria spp,Neospora spp and Sarcocystis spp. Other examples of protozoan parasitesinclude hemoflagellates, such as Leishmania spp. and Trypanosoma spp.,and other flagellated protozoans such as Giardia spp.

Plasmodium spp. include Plasmodium falciparum, Plasmodium berghei (e.g.strain ANKA), Plasmodium yoelii, Plasmodium chabaudi, Plasmodium vivax,Plasmodium knowlesi, Plasmodium ovale, Plasmodium malariae, andPlasmodium vinckei. Toxoplasma spp. include Toxoplasma gondii. Eimeriaspp. include Eimeria tenella and Eimeria acervulina. Babesia spp.include Babesia microti, Babesia divergens, and Babesia duncani.Theileria spp. include Theileria annulata, Theileria parva, Theileriaequi and Theileria orientalis. Neospora spp. include Neospora caninum.Sarcosystis spp. include Sarcocystis neurona. Leishmania spp. includeLeishmania major, Leishmania tropica, Leishmania aethiopica, Leishmaniainfantum (or Leishmania chagasi), Leishmania donovani, Leishmaniaamazonensis and Leishmania braziliensis. Trypanosoma spp. includeTrypanosoma brucei (subspecies gambiense, rhodesiense, or brucei), andTrypanosoma cruzi. Giardia spp. include Giardia lamblia (or Giardiaintestinalis).

Parasitic helminths include nematodes (e.g. hookworm, whipworm,filariid, ascarid, strongyle and trichostrongyle nematodes), such asAncyclostoma spp., Necator spp., Brugia spp., Wuchereria spp., Loa spp.,Mansonella spp., Trichinella spp., Trichuris spp., Ascaris spp.,Anisakis spp., Dracunculus spp., Strongyloides spp., Haemonchus spp.,Toxocara spp., Dictyocaulus spp., Ostertagia spp., Teladorsagia spp,Onchocerca spp. and Dirofilaria spp. Further examples includeAcanthocheilonema spp., Aelurostrongylus spp., Angiostrongylus spp.,Bunostomum spp., Dioctophyme spp., Dipetalonema spp., Lagochilascarisspp., Muellerius spp., Parailaria spp., Parascaris spp., Protostrongylusspp., Setaria spp., Stephanoflaria spp., Strongylus spp., Thelazia spp.,Capillaria spp., Chabertia spp., Cooperia spp., Enterobius spp.,Nematodirus spp., Oesophagostomum spp, and Trichostrongylus spp.

Other examples of helminthic parasites include trematodes, such asSchistosoma spp. and liver flukes such as Fasciola spp (e.g. Fasciolahepatica).

Ancyclostoma spp. include Ancylostoma duodenale, Ancylostoma ceylanicum,Ancylostoma braziliense, Ancylostoma caninum, Ancylostoma pluridentatum,and Ancylostoma tubaeforme. Necator spp. include Necator americanus.Brugia spp. include Brugia malayi, Brugia timori and Brugia pahangi.Wuchereria spp. include Wuchereria bancrofti. Loa spp. include Loa loa.Mansonella spp. include Mansonella streptocerca, Mansonella perstans andMansonella ozzardi. Trichinella spp. include Trichinella spiralis,Trichinella pseudospiralis, Trichinella nelsoni, Trichinella britovi,Trichinella murrelli, and Trichinella nativa. Trichuris spp. includeTrichuris trichiura, Trichuris muris, Trichuris campanula, Trichurissuis, Trichuris ovis and Trichuris vulpis. Ascaris spp. include Ascarislumbricoides, and Ascaris suum. Anisakis spp. include Anisakis simplex.Dracunculus spp. include Dracunculus medinensis, and Dracunculusinsignis. Strongyloides spp. include Strongyloides stercoralis andStrongyloides papillosus. Onchocerca spp. include Onchocerca volvulusand Onchocerca tubingensis. Dirofilaria spp. include Diroilaria immitisand Dirofilaria repens. Haemonchus spp. include Haemonchus contortus andHaemonchus placei. Toxocara spp. include Toxocara canis and Toxocaracati. Dictyocaulus spp. include Dictyocaulus viviparus and Dictyocaulusarnieldi. Chabertia spp. include Chabertia ovina. Cooperia spp. includeCooperia curticei and Cooperia oncophora. Nematodirus spp. includeNematodirus spathiger and Nematodirus filicollis. Oesophagostomum spp.include Oesophagostomum columbianum and Oesophagostomum venulosum.Trichostrongylus spp. include Trichostrongylus axei, Trichostrongyluscolubriformis and Trichostrongylus vitrinus. Further parasitic nematodesinclude Parastrongyloides trichosuri, Ostertagia ostertagi, Ostertagiatrifurcata, and Teladorsagia circumcincta.

Schistosoma spp. include Schistosoma mansoni, Schistosoma haematobium,Schistosoma japonicum, Schistosoma mekongi, Schistosoma bovis,Schistosoma mattheei, Schistosoma margrebowiei, Schistosoma curassoni,Schistosoma rodhaini, Schistosoma indicum, Schistosoma intercalatum,Schistosoma malayensis, Schistosoma ovuncatum, Schistosoma nasale, andSchistosoma spindale. Fasciola spp. include Fasciola hepatica, Fasciolamagna, Fasciola gigantica, Fasciola jacksoni.

Reference 46 speculatively describes therapeutic compositions to protectanimals from diseases caused by parasitic helminths which compriseparasitic helminth MIF protein, nucleic acids which hybridize understringent conditions with a Dirofilaria immitis MIF gene or anOnchocerca volvulus MIF gene, and isolated antibodies that selectivelybind to a parasitic helminth MIF protein. However, unlike the presentdisclosure, such therapeutic compositions are not substantiated withexperimental evidence which establishes that such compositions wouldhave a therapeutic effect.

Nonetheless, in some embodiments of the present invention, the parasiteis not a parasitic helminth. In some embodiments, the parasite is not aparasitic nematode. In some embodiments, the parasite is not a filariidnematode worm. In some embodiments, in particular wherein thecomposition comprises a nucleic acid comprising a sequence which encodesa parasite MIF antigen, the parasite is not Onchocerca volvulus orDirofilaria immitis. For example, in some embodiments the parasite MIFantigen (e.g. the parasite MIF antigen encoded by a nucleic acid of thecomposition) is not an Onchocerca volvulus MIF antigen or Diroflariaimmitis MIF antigen.

Reference 47 evaluated the immune response induced by DNA vaccinesexpressing Trichinella spiralis MCD-1, T. spiralis MIF or co-expressingT. spiralis MCD-1 and MIF, in BALB/c mice. Immunization with the vaccineco-expressing MCD-1 and MIF was followed by a small reduction inparasite burden but DNA vaccine expressing T. spiralis MIF did notprotect mice against T. spiralis infection. In some embodiments of thepresent invention, the parasite is not Trichinella spiralis, inparticular wherein the composition comprises a nucleic acid (and inparticular DNA) comprising a sequence which encodes a parasite MIFantigen. In some embodiments the parasite MIF antigen (e.g. the parasiteMIF antigen encoded by a nucleic acid of the composition) is not aTrichinella spiralis MIF antigen. In some embodiments, the compositionand/or the parasite MIF antigen does not comprise a multi-cystatin-likedomain protein, such as T. spiralis MCD-1.

Parasite infections may, for example, include malaria (e.g. cerebralmalaria), toxoplasmosis, coccidiosis, babesiosis, theileriosis,trypanosomiasis, leishmaniasis, filariasis, elephantiasis, trichinosis,trichuriasis, ascariasis, dracunculiasis (guinea worm disease), equineprotozoal myeloencephalitis (EPM), fasciolosis and schistosomiasis.Preferably, the methods and compositions disclosed herein are used toprovide protective immunity against malaria.

The methods and compositions disclosed herein may be used to provideprotective immunity against a single parasite infection, or co-infectionby two or more different parasites. The two or more parasites may be twoor more parasites selected from those listed herein. In someembodiments, at least one of the two or more different parasites is aPlasmodium parasite, such as a Plasmodium falciparum parasite.

Methods of Treatment and Medical Uses

The compositions disclosed herein may be used in a method for providingprotective immunity against a parasite infection in a subject (e.g. avertebrate) in need thereof, comprising the step of administering thecomposition (e.g. an immunologically effective amount of thecomposition) to the subject.

The invention also provides a composition as disclosed herein for use inproviding protective immunity against a parasite infection in a subject.

In some embodiments, the composition provides protective immunity bygenerating a protective immune response against the parasite infection(e.g. active immunization), particularly in those embodiments where thecomposition comprises a nucleic acid comprising a sequence which encodesa parasite MIF antigen or comprises a parasite MIF antigen. Protectiveimmunity may also be provided by administering to the subject anantibody which specifically binds the parasite MIF antigen (e.g. bypassive immunization). Protective immunity may also be provided byadministering to the subject parasite-responsive CD4 T cells isolatedfrom a compatible host (preferably of the same species as the subject),wherein the host has been immunized with a composition of the invention:i.e. a composition which comprises an immunologically effective amountof (i) a nucleic acid comprising a sequence which encodes a parasite MIFantigen or (ii) a parasite MIF antigen (e.g. by adoptive transfer of CD4T cells). The compatible host may have been immunized with thecomposition and exposed to the parasite or a parasite antigen to producea parasite-responsive CD4 T cell population.

Administration of a composition of the invention to a subject (orcompatible host) enables the subject (or host) to produce aparasite-responsive CD4 memory T cell population on exposure to theparasite or a parasite antigen. The parasite-responsive CD4 memory Tcell population may confer “sterilizing” immunity (i.e. completeprotective immunity) against re-infection.

Thus, also provided herein is a method of providing a subject withprotective (e.g. sterilizing) immunity against parasite re-infection.The method may comprise: (i) administering a composition of theinvention (e.g. a composition comprising a nucleic acid comprising asequence which encodes a parasite MIF antigen) to the subject, and (ii)subsequently infecting the subject with the parasite or immunizing thesubject with another parasite antigen (to produce a parasite-responsiveCD4 T cell population). In step (ii), subsequent infection of a subjectwith the parasite might be deliberate in an animal study, but in humansit is preferred that this occurs via natural infection. Thus the methodmay be applied to a subject who is likely to be exposed to infectione.g. who lives in a malarial area, or who works with malaria patients,etc. Optionally (e.g. where the subject is infected with the parasite),these methods may further comprise curing the subject of the parasiteinfection, e.g. by administration of an agent which kills or attenuatesthe parasite, as described herein.

“Protective immunity” or a “protective immune response”, as used herein,refers to immunity or eliciting an immune response against an infectiousagent (e.g. a parasite), which is exhibited by a subject, that preventsor ameliorates an infection or reduces at least one symptom thereof.Specifically, induction of protective immunity or a protective immuneresponse from administration of a composition of the invention isevident by elimination or reduction of the presence of one or moresymptoms of the parasitic infection and/or an expansion of theparasite-responsive memory T cell population. As used herein, the term“immune response” refers to both the humoral immune response and thecell-mediated immune response. Preferably, the protective immunityprovided by the invention is characterized by protective immunologicalmemory (e.g. a protective memory T cell response) against the parasite.The protective immunity may be characterized by an effectiveparasite-responsive (e.g. Plasmodium-responsive) memory T cellpopulation. In preferred embodiments, protective immunity is maintained(i.e. the protective effect does not decrease over the course of theparasite infection). Preferably, the subject can recover from theparasite infection. In preferred embodiments, treatment with acomposition of the invention as described herein provides protectiveimmunity against re-infection by the parasite. Protective immunity maybe sterilizing immunity (i.e. complete protective immunity), whereby theprotected subject can elicit an immune response which completelyeliminates the infection.

A composition of the invention may therefore treat or prevent a parasiteinfection (or a disease associated therewith). In preferred embodiments,the disease is malaria. More particularly, the disease may be cerebralmalaria.

The compositions disclosed herein may be used to induce a primary immuneresponse and/or to boost an immune response.

Where a subject is treated in accordance with the present invention, thesubject may have an expanded parasite-responsive (e.g.Plasmodium-responsive) memory T cell population relative to an untreatedsubject following parasite infection, particularly in those embodimentswhere the composition comprises a nucleic acid comprising a sequencewhich encodes a parasite MIF antigen or comprises a parasite MIFantigen. The parasite-responsive memory T cell population may compriseCD4⁺Ki67⁺ IL-7Rα⁺ T cells. For example, the parasite-responsive memory Tcells may comprise T memory cells (CD4⁺Ki67⁺ CD62L⁺ IL-7Rα⁺) and/or Teffector memory cells (CD4⁺Ki67⁺ CD62L⁻ IL-7Rα⁺). Theparasite-responsive memory T cell population may be expanded by at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50% (e.g.relative to an untreated subject) following parasite infection.

Where a subject is treated in accordance with the present invention, thesubject may have decreased IFN-γ levels relative to an untreated subjectfollowing parasite infection. For example, serum IFNγ levels may bedecreased. IFNγ production by inflammatory, terminal effector CD4 Tcells may be decreased. IFNγ levels may be assessed by specific ELISA.The decrease may be at least 10%, at least 20%, at least 30%, at least40%, at least 50%, or at least 60% relative to an untreated subjectfollowing parasite infection.

A composition of the invention may be administered to a subject to allowthe subject to develop and/or maintain sterilizing immunity to parasiteinfection. Protective immunity may be provided or augmented in animmunized subject following parasite infection or exposure to anotherparasite vaccine or antigen (e.g. an additional parasite antigen, asdefined herein).

A composition of the invention may be used in a prime-boost vaccinationregime. Protective immunity against a parasite infection according tothe invention may be provided by administering a priming vaccine,comprising a composition of the invention, followed by a boostervaccine. The booster vaccine is different from the primer vaccine andcomprises or encodes one or more additional (i.e. non-MIF) parasiteantigens, as defined herein. The booster vaccine may, for example,comprise an attenuated form of the parasite to which protective immunityis to be provided. Preferably the priming and booster vaccines areadministered less than about 16 weeks apart (e.g. less than about 8weeks, 6 weeks, 4 weeks, 2 weeks or 1 week apart). As an alternative,two vaccines may be administered within 12 hours of each other, within 6hours, within 3 hours, within 2 hours or within 1 hour of each other.

Where the composition of the invention comprises antibody whichspecifically binds to a parasite MIF antigen (anti-parasite MIFantibody), the composition may be used in a method of treating aparasite infection and/or a method of providing protective immunityagainst a parasite infection i.e. passive immunisation for therapeuticor prophylactic purposes. For example, the composition may be used in amethod of treating (i.e. ameliorating) one or more symptoms of malaria(i.e. where the parasite is Plasmodium). The symptoms may be selectedfrom headache, fever, shivering, joint pain, vomiting, hemolytic anemia,jaundice, hemoglobinuria, retinal damage, convulsions, encephalopathy,or one or more neurological symptoms, including abnormal posturing,nystagmus, conjugate gaze palsy (failure of the eyes to turn together inthe same direction), opisthotonus, seizures, or coma. The malaria may becerebral malaria.

In some embodiments, the anti-parasite MIF antibody is administered toan infected subject. The subject may be a recently-infected subject. Forexample, the antibody may be administered within 2 weeks of infection(e.g. within 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3days, 2 days, 24 hrs). Alternatively, the subject may be at risk ofinfection. For example, the subject may be at risk of infection within 2weeks of administration of the antibody (e.g. within 10 days, 9 days, 8days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 24 hrs).

In certain embodiments, the subject is a vertebrate, e.g., a mammal,such as a human or a veterinary mammal (e.g. cat, dog, horse, cow,sheep, cattle, deer, goat, or pig) as the parasites covered herein maybe problematic across a wide range of species.

The compositions of the invention can be formulated as vaccinecompositions. Vaccines according to the invention may either beprophylactic (i.e. to prevent infection) or therapeutic (i.e. to treatinfection), but will typically be prophylactic.

Compositions of the invention may be used to treat both children andadults. Where the vaccine is for prophylactic use, the human ispreferably a child (e.g. a toddler or infant) or a teenager; where thevaccine is for therapeutic use, the human is preferably a teenager or anadult. A vaccine intended for children may also be administered toadults e.g. to assess safety, dosage, immunogenicity, etc.

Thus a human subject may be less than 1 year old, less than 5 years old,1-5 years old, 5-15 years old, 15-55 years old, or at least 55 yearsold. Preferred patients for receiving the compositions are the elderly(e.g. ≥50 years old, ≥60 years old, and preferably ≥65 years), the young(e.g. ≤5 years old), hospitalised patients, healthcare workers, armedservice and military personnel, pregnant women, the chronically ill, orimmunodeficient patients. The compositions are not suitable solely forthese groups, however, and may be used more generally in a population.

The subject may have previously been infected with and/or mounted animmune response against the parasite of interest. For example, thesubject may have previously mounted an immune response against theparasite MIF antigen of interest. Alternatively, the subject may beimmunologically naïve with respect to the parasite and/or the parasiteMIF antigen.

By “immunologically effective amount”, it is meant that theadministration of that amount to a subject, either in a single dose oras part of a series, is effective for treatment or prevention of aparasite infection. This amount varies depending upon the health andphysical condition of the individual to be treated, age, the taxonomicgroup of individual to be treated (e.g. human, non-human primate, etc.),the capacity of the individual's immune system to synthesise antibodies,the degree of protection desired, the formulation of the composition orvaccine, the treating doctor's assessment of the medical situation, theseverity of the disease, the potency of the compound administered, themode of administration, and other relevant factors. It is expected thatthe amount will fall in a relatively broad range that can be determinedthrough routine trials.

A dose of a nucleic acid (e.g. a nucleic acid-based vaccine) may have≤100 μg nucleic acid; e.g. from 10-100 μg, such as about 10 μg, 25 μg,50 μg, 75 μg or 100 μg, but expression can be seen at much lower levels;e.g. using 1 μg/dose, ≤100 ng/dose, ≤10 ng/dose, ≤1 ng/dose, etc.Similarly, a dose of a protein antigen may have ≤100 μg protein; e.g.from 10-100 μg, such as about 10 μg, 25 μg, 50 μg, 75 μg or 100 μg. Adose of an antibody may have ≤1000 mg antibody or ≤500 mg antibody; e.g.from 1-1000 mg or 1-500 mg, such as about 10 mg, 20 mg, 50 mg, 100 mg,200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg or 900 mg. Theantibody may be administered in a dose of about 0.1-10 mg/kg bodyweight.

Compositions of the invention will generally be administered directly toa subject. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,intradermally, or to the interstitial space of a tissue). Alternativedelivery routes include rectal, oral (e.g. tablet, spray), buccal,sublingual, vaginal, topical, transdermal or transcutaneous, intranasal,ocular, aural, pulmonary or other mucosal administration. Intradermaland intramuscular administration are two preferred routes. Injection maybe via a needle (e.g. a hypodermic needle), but needle-free injectionmay alternatively be used. A typical intramuscular dose is 0.5 ml.

One way of checking efficacy of therapeutic treatment involvesmonitoring parasite infection after administration of the compositionsdisclosed herein. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses, systemically (such as monitoringthe level of IgG1 and IgG2a production) and/or mucosally (such asmonitoring the level of IgA production), against the antigen. Typically,antigen-specific serum antibody responses are determinedpost-immunization but pre-challenge whereas antigen-specific mucosalantibody responses are determined post-immunization and post-challenge.

Another way of assessing the immunogenicity of the compositionsdisclosed herein is to express the parasite MIF antigen recombinantlyfor screening patient sera or mucosal secretions by immunoblot and/ormicroarrays. A positive reaction between the parasite MIF antigen andthe patient sample indicates that the patient has mounted an immuneresponse to the antigen. This method may also be used to identifyimmunodominant antigens and/or epitopes within protein antigens.

The efficacy of the compositions can also be determined in vivo bychallenging appropriate animal models of the parasite infection ofinterest.

Dosage can be by a single dose schedule or a multiple dose schedule(i.e. two or more doses). Multiple doses may be used in a primaryimmunisation schedule and/or in a booster immunisation schedule. In amultiple dose schedule the various doses may be given by the same ordifferent routes e.g. a parenteral prime and mucosal boost, a mucosalprime and parenteral boost, etc. Multiple doses will typically beadministered at least 1 week apart (e.g. about 2 weeks, about 3 weeks,about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12weeks, about 16 weeks, etc.). In one embodiment, two or more doses areadministered about 3 weeks apart.

Where multiple doses (i.e. two or more doses) of a composition of theinvention are administered to a subject, the composition used for eachdose may be independently selected from a composition which comprises:(i) a nucleic acid comprising a sequence which encodes a parasite MIFantigen; (ii) a parasite MIF antigen; and (iii) an antibody whichspecifically binds to a parasite MIF antigen. The compositionadministered in a first dose (the first dose composition) may be acomposition which comprises any of (i), (ii) and/or (iii) and thecomposition administered in a second dose (the second dose composition)may be a composition which comprises any of (i), (ii) and/or (iii). Thecomposition in each of the two or more doses may be the same (e.g. afirst dose of (i) followed by a second dose of (i), or a first dose (ii)followed by a second dose of (ii)) or may be different (e.g. a firstdose of (i) followed by a second dose of (ii), or a first dose of (ii)followed by a second dose of (i)). In each of (i), (ii) and (iii), ineach dose, the parasite MIF antigen may be the same parasite MIFantigen.

Pharmaceutical Compositions

The invention provides compositions comprising (i) a nucleic acidcomprising a sequence which encodes a parasite MIF antigen; (ii) aparasite MIF antigen; or (iii) an antibody which specifically binds to aparasite MIF antigen. The composition may be a pharmaceuticalcomposition, for example a vaccine composition. Accordingly, thecomposition may also comprise a pharmaceutically acceptable carrier.Where the composition comprises a nucleic acid comprising a sequencewhich encodes a parasite MIF antigen (e.g. nucleic acid-based vaccine),the composition may also comprise a delivery system, as describedherein.

A “pharmaceutically acceptable carrier” includes any carrier that doesnot itself induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolised macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,sucrose, trehalose, lactose, and lipid aggregates (such as oil dropletsor liposomes). Such carriers are well known to those of ordinary skillin the art. The compositions may also contain a pharmaceuticallyacceptable diluent, such as water, saline, glycerol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present. Sterilepyrogen-free, phosphate-buffered physiologic saline is a typicalcarrier. A thorough discussion of pharmaceutically acceptable excipientsis available in ref. 48.

Pharmaceutical compositions may include the particles in plain water(e.g. w.f.i.) or in a buffer e.g. a phosphate buffer, a Tris buffer, aborate buffer, a succinate buffer, a histidine buffer, or a citratebuffer. Buffer salts will typically be included in the 5-20 mM range.

Pharmaceutical compositions may have a pH between 5.0 and 9.5 e.g.between 6.0 and 8.0.

Compositions may include sodium salts (e.g. sodium chloride) to givetonicity. A concentration of 10±2 mg/ml NaCl is typical e.g. about 9mg/ml.

Compositions may include metal ion chelators. These can prolong RNAstability by removing ions which can accelerate phosphodiesterhydrolysis. Thus a composition may include one or more of EDTA, EGTA,BAPTA, pentetic acid, etc. Such chelators are typically present atbetween 10-500 μM e.g. 0.1 mM. A citrate salt, such as sodium citrate,can also act as a chelator, while advantageously also providingbuffering activity.

Pharmaceutical compositions may have an osmolality of between 200mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, or between290-310 mOsm/kg.

Pharmaceutical compositions may include one or more preservatives, suchas thiomersal or 2-phenoxyethanol. Mercury-free compositions arepreferred, and preservative-free vaccines can be prepared.

Pharmaceutical compositions are preferably sterile.

Pharmaceutical compositions may be non-pyrogenic e.g. containing ≤1 EU(endotoxin unit, a standard measure) per dose, and preferably ≤0.1 EUper dose.

Pharmaceutical compositions may be gluten free.

Pharmaceutical compositions may be prepared in unit dose form. In someembodiments a unit dose may have a volume of between 0.1-1.0 ml e.g.about 0.5 ml.

The compositions may be prepared as injectables, either as solutions orsuspensions. The composition may be prepared for pulmonaryadministration e.g. by an inhaler, using a fine spray. The compositionmay be prepared for nasal, aural or ocular administration e.g. as sprayor drops. Injectables for intramuscular administration are typical.

The invention also provides a delivery device (e.g. syringe, nebuliser,sprayer, inhaler, dermal patch, etc.) containing a pharmaceuticalcomposition of the invention. This device can be used to administer thecomposition to a subject (e.g. a vertebrate subject).

A composition of the present disclosure may also comprise, or beadministered in conjunction with, one or more adjuvants (e.g. vaccineadjuvants), in particular where the composition comprises animmunologically effective amount of a parasite MIF antigen.

Adjuvants which may be used in compositions of the invention include,but are not limited to:

(A) Mineral-containing compositions, for example aluminium and calciumsalts, such as aluminium phosphates. (B) Oil emulsions, for examplesqualene-in-water emulsions, such as MF59 or AS03. Complete Freund'sadjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used.(C) Saponin formulations. (D) Virosomes and virus-like particles (VLPs).(E) Bacterial or microbial derivatives such as non-toxic derivatives ofenterobacterial lipopolysaccharide (LPS), Lipid A derivatives,immunostimulatory oligonucleotides and ADP-ribosylating toxins anddetoxified derivatives thereof. (F) Human immunomodulators, for examplecytokines, such as interleukins, interferons, macrophage colonystimulating factor, and tumor necrosis factor. (G) Bioadhesives andmucoadhesives, such as esterified hyaluronic acid microspheres,cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol,polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. (H)Microparticles, for example particles of ˜100 nm to ˜150 μm in diameter,more preferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500nm to ˜10 μm in diameter) formed from materials that are biodegradableand non-toxic (e.g. a poly(a-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB). (I) Liposomes.(J) Polyoxyethylene ether and polyoxyethylene ester formulations. (K)Polyphosphazene (PCPP). (L) Muramyl peptides. (M) Imidazoquinolonecompounds, for example Imiquamod and its homologues.

The use of an aluminium hydroxide or aluminium phosphate adjuvant isparticularly preferred, and antigens are generally adsorbed to thesesalts. Alternatively, MF59, AS01 or AS03 may be used as the adjuvant.

Exemplary adjuvants also include human TLR7 agonists, such as a compoundof formula (K). These agonists are discussed in detail in reference 49:

wherein:

R¹ is H, C₁-C₆alkyl, —C(R⁵)₂OH, -L¹R⁵, -L¹R⁶, -L²R⁵, -L²R⁶, -OL²R⁵, or-OL²R⁶;

L¹ is —C(O)— or —O—;

L² is C₁-C₆alkylene, C₂-C₆alkenylene, arylene, heteroarylene or—((CR⁴R⁴)_(p)O)(CH₂)_(p)—, wherein the C₁-C₆alkylene and C₂-C₆alkenyleneof L² are optionally substituted with 1 to 4 fluoro groups;

each L³ is independently selected from C₁-C₆alkylene and—((CR⁴R⁴)_(p)O)(CH₂)_(p)—, wherein the C₁-C₆alkylene of L³ is optionallysubstituted with 1 to 4 fluoro groups;

L⁴ is arylene or heteroarylene;

R² is H or C₁-C₆alkyl;

R³ is selected from C₁-C₄alkyl, -L³R⁵, -L¹R⁵, -L³R⁷, -L³L⁴L³R⁷, -L³L⁴R⁵,-L³L⁴L³R⁵, - OL³R⁵, —OL³R⁷, —OL³L⁴R⁷, —OL³L⁴L³R⁷, —OR⁸, —OL³L⁴R⁵,—OL³L⁴L³R⁵ and —C(R⁵)₂OH;

each R⁴ is independently selected from H and fluoro;

R⁵ is —P(O)(OR⁹)₂,

R⁶ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰;

R⁷ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰;

R⁸ is H or C₁-C₄alkyl;

each R⁹ is independently selected from H and C₁-C₆alkyl;

R¹⁰ is H or C₁-C₄alkyl;

each p is independently selected from 1, 2, 3, 4, 5 and 6, and

q is 1, 2, 3 or 4.

A TLR7 agonist, such as a TLR7 agonist of formula (K), may be adsorbedto an insoluble aluminium salt (e.g. to form an adsorbed complex foradjuvanting immunogens). Useful aluminium salts include, but are notlimited to, aluminium hydroxide and aluminium phosphate adjuvants.Aluminium salts which include hydroxide ions are preferred for use withthe invention as these hydroxide ions can readily undergo ligandexchange with compounds of formula (K). Thus preferred salts foradsorption of TLR agonists are aluminium hydroxide and/or aluminiumhydroxyphosphate. These have surface hydroxyl moieties which can readilyundergo ligand exchange with phosphorus-containing groups (e.g.phosphates, phosphonates) to provide stable adsorption. In an exemplaryembodiment, an aluminium hydroxide adjuvant is used. Alternatively, aTLR7 agonist, such as a TLR7 agonist of formula (K) may not be adsorbedto an insoluble aluminium salt.

Phosphorous-containing adjuvants used with the invention may exist in anumber of protonated and deprotonated forms depending on the pH of thesurrounding environment, for example the pH of the solvent in which theyare dissolved. Therefore, although a particular form may be illustrated,it is intended that these illustrations are merely representative andnot limiting to a specific protonated or deprotonated form. For example,in the case of a phosphate group, this has been illustrated as—OP(O)(OH)₂ but the definition includes the protonated forms[OP(O)(OH₂)(OH)]⁺ and —[OP(O)(OH)₂]²⁺ that may exist in acidicconditions and the deprotonated forms —[OP(O)(OH)(O)]⁻ and [OP(O)(O)₂]²⁻that may exist in basic conditions

Combinations of one or more of the adjuvants identified above may alsobe used with the invention.

Sequence Identity

Identity or homology with respect to a sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the reference amino acid sequence after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity.

Sequence identity can be determined by standard methods that arecommonly used to compare the similarity in position of the amino acidsof two polypeptides. Using a computer program such as BLAST or FASTA,two polypeptides are aligned for optimal matching of their respectiveamino acids (either along the full length of one or both sequences oralong a pre-determined portion of one or both sequences). The programsprovide a default opening penalty and a default gap penalty, and ascoring matrix such as PAM 250 [a standard scoring matrix; see Dayhoffet al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3(1978)] can be used in conjunction with the computer program. Forexample, the percent identity can then be calculated as: the totalnumber of identical matches multiplied by 100 and then divided by thesum of the length of the longer sequence within the matched span and thenumber of gaps introduced into the shorter sequences in order to alignthe two sequences.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x+10%.

Where the present disclosure refers to a sequence by reference to aUniProt or Genbank accession code, the sequence referred to is thecurrent version at the filing date of the present application.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1D show malaria-specific T cell responses in mice immunizedwith: RNA encoding PbMIF in a CNE delivery vehicle (PbMIF-CNE); RNAencoding GFP in a CNE delivery vehicle (GFP-CNE); or mice immunized withPbMIF protein with Freund's adjuvant (PbMIF-FA); at 7 days afterPlasmodium infection on day 40 post-immunization. *P<0.05. FIG. 1A shows% CD4⁺ Ki67⁺. FIG. 1B shows B-bet MFI. FIG. 1C shows % PbA respondingCD4 T cells. FIG. 1D shows IGN-γ MFI.

FIGS. 2A-B showneutralization activity of PbMIF immunization on (FIG.2A) Plasmodium parasitemia and (FIG. 2B) spleen parasite content at 7days post-infection. *P<0.05

FIG. 3 shows mean serum anti-PbMIF titers (OD_(450 nm)) at day 14 (2weeks after first immunization) for mice immunized with RNA/PMIF CNE orRNA/GFP CNE. Mean titers are expressed as endpoint titers; thereciprocal dilution that yields background OD_(450 nm). ****P<0.0001Mann-Whitney U, n=15 per group.

FIG. 4 shows mean serum anti-PbMIF titers (OD_(450 nm)) at day 35 (2weeks after second immunization) for mice immunized with RNA/PMIF CNE orRNA/GFP CNE. Mean titers are expressed as in FIG. 3. ****P<0.0001Mann-Whitney U, n=15 per group.

FIGS. 5A-C show: (FIG. 5A) a scheme for RNA/PbMIF CNE and RNA/GFP CNEvaccination, first parasite challenge, cure (CQ), and second parasitechallenge; (FIG. 5B) Plasmodium parasitemia over 7 days following firstparasite challenge (days 35 to 42); and (FIG. 5C) serum IFNγ levels onday 5 post-infection. *P<0.05, **P<0.01 Mann-Whitney U, n=15 per group.

FIG. 6A shows mean serum anti-PbMIF titers (OD_(450 nm)) at day 59 (2.5weeks after first infection) for mice immunized with RNA/PMIF CNE orRNA/GFP CNE (****P<0.0001 Mann-Whitney U, n=15 per group). FIG. 6B showsmean anti-Plasmodium IgG responses at day 59 (***P<0.01 Mann-Whitney U,n=15 per group). Mean titers are expressed as in FIG. 3.

FIG. 7 shows Plasmodium parasitemia in mice immunized with RNA/PMIF CNEor RNA/GFP CNE over 14 days following second challenge (days 59-73).*P<0.05, **P<0.01 Mann-Whitney U, n=9 total (4×RNA/GFP CNE control,5×RNA/PMIF CNE).

FIGS. 8A-C show the effects of RNA/PMIF CNE versus or RNA/GFP CNEimmunization on T cell phenotype. T cells responding to P. berghei(CD4+Ki67^(hi)) were divided into subsets: Tmem (memory) CD62L⁺ IL-7Rα⁺;Tem (effector memory) CD62L⁻ IL-7Rα⁺; and Teff (effector) CD62L⁻IL-7Rα⁻. FIG. 8A shows percentages of individual T cell phenotypes at 7days after first infection. FIG. 8B shows percentages of individual Tcell phenotypes at 7 days after second infection. FIG. 8C showspercentages of Tmem cells which are PD-I⁺ (indicating T cellexhaustion). *P<0.05, **P<0.01 Mann-Whitney U.

FIG. 9 shows Plasmodium parasitemia in mice receiving passive transferof a polyclonal anti-PbMIF antibody (IgG) compared to mice receivingcontrol IgG from a non-immunized rabbit (Ctrl IgG) at day 7post-infection. Each data point is a single mouse. P=0.0005 by t-test.

FIGS. 10A-E show that passive transfer of IgG from PbMIF immunized andblood-stage infected mice provides partial protection in both BALB/c andcerebral malaria-sensitive C57BL/6 Mice. (FIG. 10A) IgG isolated fromGFP (control) or PbMIF immunized and PbA-infected mice was administeredi.p. to naïve BALB/c or C57BL/6 mice and followed by PbA infection.(FIG. 10B) Parasitemia and (FIG. 10C) Kaplan-Meyer survival analysis ofBALB/c mice administered immune IgG. p values were generated using aLong-rank test and data are from two independent experiments with 5 miceper group (*p<0.05, **p<0.01). (FIG. 10D) Kaplan-Meyer survival plotsand (FIG. 10E) ECM (Experimental Cerebral Malaria) score of C57BL/6 miceadministered immune IgG and infected with PbA. Statistical p values weregenerated using a Long-rank test (**p<0.01) and data are representativeof two independent experiments n=10 mice per group.

FIGS. 11A-G shows that adoptively transferred CD4 T cells from PbMIFimmunized mice confer protection to homologous challenge. (FIG. 11A)Immunized CD45.2 BALB/c mice were infected with 10⁶ PbA-iRBCs andtreated with chloroquine on days 7-12. Four weeks later, the mice werere-infected with PbA and splenocytes isolated 7 days after infection,incubated with chloroquine to eliminate residual Plasmodium, and labeledwith CFSE. Purified CD4 T cells (2×10⁷) then were transferred into naïveCD45.1 BALB/c mice and infected 3 days later with 10⁶ PbA-iRBCs. (FIG.11B) Parasitemia in mice adoptively transferred with CD4 T cells fromGFP (◯) or PbMIF (●) immunized mice (*p<0.05, #p<0.001, by two-wayANOVA). The graph shows % parasitemia against days post-infection. (FIG.11C) Representative CFSE dilution histogram of adoptively transferred(CD45.2) CD4 T cells from GFP or PbMIF immunized donors, (FIG. 11D)enumeration of recovered CD45.2 CD4 T cells, and (FIG. 11E)proliferation response of transferred CD4 T cells in CD45.1 recipients 7days after infection. (FIG. 1F) Percent of proliferating CD45.2 CD4 Tcells (CFSE^(lo)) producing IFNγ after stimulation ex vivo with PbA-iRBClysates. (FIG. 11G) Mean fluorescence intensity of PD-1 inPbA-responsive CD45.2 CD4 T cells (CFSE^(lo)) from GFP (control) orPbMIF immunized donors. Results are from two separate experiments with 4mice per group (*p<0.05, **p<0.01 by Mann-Whitney test.).

MODES FOR CARRYING OUT THE INVENTION Example 1: Immunization Using P.berghei MIF, Followed by Parasite Challenge

Groups of 5 female BALB/c mice aged 8-10 weeks were immunized with: (1)RNA encoding P. berghei MIF (PbMIF) in an LNP delivery vehicle (RV01,see references 34 and 36); (2) RNA encoding PbMIF in a CNE deliveryvehicle (comprising squalene and DOTAP, as described in reference 35);(3) RNA encoding GFP in a CNE delivery vehicle; or (4) intraperitoneal(i.p.) injection of PbMIF protein with Freund's adjuvant, as set out inTable 1. Immunizations were carried out on day 0 and day 21.

TABLE 1 Delivery # animals/ Group Antigen system Adjuvant Dose group 1RNA/PbMIF LNP — 1 μg 5 2 RNA/PbMIF CNE — 15 μg 5 3 RNA/GFP CNE — 15 μg 54 PbMIF i.p. FCA/FIA 10/5 μg 5 Protein

Blood samples were taken from immunized mice on day 14 and day 35 (14days after boosting) and total serum anti-PbMIF IgG titers were measuredby anti-PbMIF ELISA assay. Immunized mice were challenged on day 38-40by i.p. injection of 10⁶ P. berghei ANKA (PbA)-infected red blood cells(RBCs). Mouse weights over time (from day 0 to day 40) and clinicalappearance were also assessed for the mice in each experimental group.

Serum Anti-PbMIF IgG Titers from Immunized Mice and Tolerability to theVaccine

IgG titers were measured 14 days after the first immunization and 14days after the second boosting immunization. Anti-PbMIF ELISA assayswere performed as follows: 96 well plates were coated overnight with 100ng/ml of recombinant PbMIF. After blocking for 1 hr at room temperature,serial dilutions of sera were incubated for 2 hrs and total bound IgGwas detected with a rabbit anti-mouse IgG coupled to horseradishperoxidase (HRP). 3,3′,5,5′-Tetramethylbenzidine (TMB) was used assubstrate. The reaction was stopped with acid and the OD readingperformed at 450 nm.

Immunization with PbMIF self-replicating RNA vaccine or PbMIF protein(Groups 1, 2 and 4) elicited primary and secondary humoral antibodyresponses to PbMIF. No significant responses were observed in controlmice treated with RNA/GFP CNE (Group 3). Secondary responses wereobserved in 80% of mice treated with RNA/PMIF CNE (Group 2) and 60% ofmice treated with RNA/PMIF LNP (Group 1), with comparable titers.Primary responses in Groups 1 and 2 were ˜100-fold less and secondaryresponses in Groups 1 and 2 were ˜1000-fold less than mice treated withPMIF protein FCA/FIA (Group 4).

Mice tolerated immunization well, with no changes in clinical appearance(clinical observation q3d) and no reductions in weight among thedifferent experimental groups from day 0 to day 40.

Impact of PbMIF Immunization on Malaria-Specific T Cell Responses

Groups 2, 3 and 4 were selected for study. Immunized mice werechallenged on day 40 by i.p. injection of 10⁶ PbA-infected RBCs. On day7 post-infection, splenocytes were isolated and stimulated ex vivo byculturing with infected red blood cell lysates in the presence ofanti-CD3/CD28 beads and brefeldin A for 6 hrs. Intracellular cytokinestaining was then performed. The following antibodies were used to studyCD4 T cell IFN-γ production, T cell activation (CD11a) and T celldifferentiation (T-bet): Ki67 FITC, CD45.2 PerCP-Cy5.5, IFNγ PE-Cy7, CD4Alexa 700, CD11a eFluor 405, T-bet Alexa 647 (Life Technologies).PbA-responsive CD4 T cells are defined as CD45.2+, Ki67+, CD4+. Stainedcells were analysed by flow cytometry.

On day 7 post-infection, PbMIF immunized mice (Groups 2 and 4) showed astronger T cell proliferative response to P. berghei parasites (higherCD4+Ki67+ cells) a stronger memory CD4 T cell response to parasites, asindicated by lower CD11a and lower T-bet (FIGS. 1A and 1B) and fewerinflammatory, terminal effector IFN-γ producing T cells (FIGS. 1C and1D) than control mice (Group 3).

For example, flow cytometry showed that 5.26% of splenic CD4 T cellsfrom RNA/PMIF CNE (Group 2) immunized mice were inflammatory, terminaleffector IFN-γ-producing CD4 T cells (labelled with antiCD4 and stainedfor IFN-γ with an anti-IFN-γ antibody), compared to 11.1% of splenic CD4T cells from control RNA/GFP CNE (Group 3) immunized mice (using pooledcells from 5 mice per group).

Further studies have shown a >65% increase in the number ofPlasmodium-responsive memory CD4 T cells (CD62L+IL7Rα+) and effectormemory CD4 T cell precursors (CD62L-IL7Rα+), as well as a 20% reductionin the expression of the exhaustion marker PD-1 in PbMIF-CNE immunizedmice compared to GFP-CNE control mice at day 7 post-infection (n=5 pergroup, based on two separate experiments), suggesting a relativepreservation of the memory response in the PbMIF immunized mice versusthe control group. It has also been shown that, at day 7 post-infection,the number of Plasmodium-responsive follicular helper CD4 T cells(T_(FH) cells—CD49d^(hi)CD11a^(hi) CXCR5^(hi) CD4) was 50% greater inPbMIF-CNE immunized mice compared to GFP-CNE control mice. Consistentwith this observed increase in the population of T_(FH) cells, there wasa corresponding enhancement in splenic B cell numbers, with a 30%increase in (CD19⁺B220⁺) B cells and a greater than doubling of the Bcell plasmablast population (CD19^(lo) B220^(lo)CD138^(hi)IgD⁻). Thus,PbMIF immunization is associated with an improvement in the host T_(FH)and B cell responses.

Assessment of the Neutralization Activity of PbMIF RNA Vaccinations onP. berghei Parasitemia and Spleen Parasite Content

On day 7 post-infection, parasitemia was also studied in immunized,challenged mice from Groups 2 and 3. Parasite burden was measured byquantitative PCR detection of P. berghei 18S rRNA copies/L of peripheralblood, and splenic parasite burden was measured by expression of 18SrRNA relative to host GAPDH.

As shown in FIG. 2, PbMIF-CNE immunization was associated with asignificant reduction in parasitemia (30%) and spleen parasite contentupon challenge with lethal P. berghei infection.

Parasitemia and Survival During First Infection

In a separate experiment, BALB/c mice previously immunized with theRNA/PbMIF-CNE vaccine or GFP-CNE were infected with PbA and theparasitemia followed by FACS. Parasitemia was significantly reduced inthe RNA/PbMIF-CNE-immunized mice compared to GFP-CNE-immunized controlmice. While there was no initial difference in parasitemia between thetwo groups, there was a more rapid increase in parasitemia after day 5in the control (GFP-CNE) group, which became moribund on day 21. Bycontrast, the PbMIF immunized mice showed better control of parasitemiaduring the first 15 days of infection. Survival was also monitored forup to 30 days post-infection and RNA/PbMIF-CNE-immunized mice showed a37% prolongation in mean survival time compared to GFP-CNE-immunizedcontrol mice.

Conclusions (1)

PbMIF protein and self-replicating RNA vaccines are well-tolerated andproduce a primary and secondary humoral antibody response in BALB/cmice.

RNA/PbMIF immunization (with CNE) neutralized Plasmodium PbMIF activity,and enhanced CD4 T cell memory differentiation. In addition, theneutralization of PbMIF activity significantly reduced parasitemia andparasite content of spleens and significantly improved mean survivaltimes in infected mice. Thus, PbMIF immunization confers at leastpartial protection to first challenge infection.

Example 2: Immunization Using P. berghei MIF in CNE RNA DeliveryVehicle, Followed by Parasite Challenge, Cure and Re-Challenge

Example 2 differs from Example 1 in the addition of a first parasitechallenge, followed by cure to expand the Plasmodium-specific memory Tcell population.

Groups of 15 female BALB/c mice aged 8-10 weeks were immunized with: (1)RNA encoding PbMIF in a CNE delivery vehicle; and (2) RNA encoding GFPin a CNE delivery vehicle, as set out in Table 2. Immunizations werecarried out on day 0 and day 21.

TABLE 2 Delivery # animals/ Group Antigen system Adjuvant Dose group 1RNA/PbMIF CNE — 15 μg 15 2 RNA/GFP CNE — 15 μg 15

Blood samples were taken from immunized mice on days 14 and 35 and totalserum anti-PbMIF IgG titers were measured by anti-PbMIF ELISA assay.Immunized mice were challenged on day 35 by i.p. injection of 10⁶PbA-infected RBCs. This was followed by cure with chloroquine (CQ) (50mg/kg/day) on days 7-10 post-challenge (days 42-45).

Readouts following first challenge:

-   -   Parasitemia; days 3, 5 and 7 post-challenge (days 38, 40, 42).    -   Total serum anti-PbMIF Ig and total anti-Plasmodium Ig after CQ        cure (before 2^(n)a challenge, day 59).

Immunized mice were re-challenged with P. berghei on day 59 andinfection was followed for 7 or 14 days post-re-challenge. 5 mice/groupwere euthanized at day 4 or 7 and the remaining mice were monitored forparasitemia.

Readouts:

-   -   Parasitemia; days 5, 8, 11 and 14 post-challenge (days 64, 67,        70, 73).    -   T cell phenotypes (day 66).        Serum Anti-PbMIF IgG Titers on Day 14 (2 Weeks after First        Immunization) and Day 35 (2 Weeks after Second Immunization)

Serum IgG titers were measured by anti-PbMIF ELISA on day 14, followingthe first immunization and day 35, following the second immunization.Anti-PbMIF ELISA assays were performed as in Example 1.

Serum anti-PbMIF IgG titers were measured for each mouse at day 14. Ananti-PbMIF IgG response (˜1/2500) was observed in 50% of PbMIF-immunizedmice in Group 1 after the first immunization. Mean titers at day 14 areshown in FIG. 3.

Also, serum anti-PbMIF IgG titers were measured for each mouse at day35. An increase of the anti-PbMIF IgG titers was observed in 95% ofPbMIF-immunized mice in Group 1 after the second immunization in thePbMIF-CNE group and the titers are 5-fold higher compared to those atday 14 (˜1/14,500).

Challenge Infection on Days 35-42 to Expand Plasmodium-Specific Memory TCells

Parasitemia was assessed on days 3, 5, and 7 following first challenge(days 35-42) as described in Example 1. Serum IFNγ was also assessed byspecific ELISA on day 5 post-infection.

As shown in FIG. 5B, a detectable (0.5 log) difference in parasitemiawas observed at 7 days following first Plasmodium infection. This effectis associated with reduced circulating IFNγ that is consistent withreduced levels of a highly inflammatory, IFNγ+ T cell response (FIG.5C).

Serum Anti-PbMIF and Anti-Plasmodium IgG Titers on Day 59, 2.5 Weeksafter the First Infection

Serum anti-PbMIF IgG titers were measured for each mouse at day 59.Plasmodium infection increased the titers of anti-PbMIF Ig by 10-fold(FIG. 6A) in RNA/PbMIF versus RNA/GFP mice. Also, serum anti-PlasmodiumIgG titers were measured at day 59 by anti-mouse IgG ELISA onparasitized red cell lysates. Plasmodium infection increasedanti-Plasmodium Ig titers by 5-fold (FIG. 6B) in RNA/PbMIF versusRNA/GFP mice.

Parasitemia Following Re-Challenge with Plasmodium on Day 59

Parasitemia was assessed on days 5, 8, 11 and 14 following secondchallenge (days 59-73) as described in Example 1.

As shown in FIG. 7, RNA/PbMIF immunization markedly reduced parasitemia(5 log at day 14 post-infection) after Plasmodium re-challenge on day59.

Assessment of RNA/PbMIF Effects on Plasmodium-Specific T Cell Phenotype

Mice were euthanized at day 7 after second infection and the T cellresponse and phenotype analysed along with the cytokine production.Splenic cells were isolated from 4-5 mice per experimental group andanalysed for CD62L and IL-7Rα staining. CD62L and IL-7Rα identifydifferent T cell subsets responding to P. berghei (CD4⁺Ki67^(hi)). Theresults are summarized in Table 3.

TABLE 3 Tmem: CD4+ P. berghei-responding T memory Antigen cellsphenotyped as CD62L+IL-7Rα+ PbMIF-CNE 29.5% (Group 1) GFP-CNE 17.9%(Group 2 - control)

Thus, PbMIF neutralization increases the pool of P. berghei-respondingCD4+ memory T cells.

Impact of RNA/PbMIF Vaccine on Plasmodium-Specific T Cells

T cell phenotypes observed after first Plasmodium infection (day 7post-infection, data from study in Example 1) (FIG. 8A) were comparedwith T cell phenotypes observed after second Plasmodium infection (day7) (FIG. 8B).

As can be seen in FIGS. 8A and 8B, the RNA/PbMIF-CNE vaccine promotesthe development of Plasmodium-specific Tmem cells (CD62L IL-7Rac) duringa first infection by P. berghei and these Tmem cells increase furtherduring second infection.

Furthermore, T cell exhaustion, as indicated by PD-1-expressingPlasmodium-specific Tmem cells, was reduced in RNA/PbMIF-immunized miceversus RNA/GFP immunized mice (FIG. 8C).

T Cell Cytokine Production by Plasmodium-Responsive T Cell Subsets

IFNγ production by Tem (short-lived effector memory cells) is reduced inPbMIF immunized animals. This may reflect a less inflammatory (and moreeffective) Plasmodium-specific T cell response. No evident changes inTNF producing T cells were noted. (Data not shown)

Conclusions (2)

Self-replicating RNA vaccines are well-tolerated and produce stronghumoral responses to Plasmodium MIF (PbMIF). First and secondimmunizations induced a response in 50% and 95% of immunized mice,respectively, with a much higher titer after the second immunization,and again after PbA infection.

RNA/PbMIF (CNE) immunization neutralized Plasmodium PbMIF activity, asevidenced by enhancement in CD4 T cell memory differentiation. Theeffects of this neutralization are:

-   -   Higher Tmem cell numbers, and a stronger humoral anti-Plasmodium        antibody response. After primary challenge with P. berghei,        there is a measurable and significant decrease in parasitemia        (0.5 log).    -   Re-challenge after cure of primary infection results in a        further expansion of Tmem numbers and a significant reduction in        parasitemia (5 log).

Immunization with RNA/PbMIF allows for the development of memory T cellsand provides significant protection to malaria re-challenge. RNA/PbMIFmay be a viable vaccine candidate, either as a stand-alone or incombination with other Plasmodium vaccine candidates, where it would actto promote long-lasting memory T cell responses.

Example 3: Passive Transfer of a Polyclonal Anti-PBMIF Antibody

IgG was purified from a rabbit immunized with recombinant PbMIF(Anti-PbMIF IgG) or from a non-immunized rabbit (Ctrl IgG) and 200 μginjected i.p. into C57BL/6 mice at −6 hrs, 24 hrs, 48 hrs, and 72 hrsafter infection with 1×10⁶ P. berghei parasitized red cells. Parasitemiawas enumerated by quantitative PCR of blood at day 7 post-infection.

As shown in FIG. 9, passive transfer of the anti-PbMIF antibodysignificantly reduces parasitemia in the P. berghei infection mousemodel of malaria.

In a further study, 200 μg of IgG purified from serum by protein-Gchromatography (Pierce) from GFP (control) or PbMIF immunized andPbA-infected mice was administered i.p. to naïve BALB/c or C57BL/6(cerebral malaria-sensitive) mice and followed by PbA infection (FIG.10A). I.p. administration was on days −1, 1, 2, and 3 post-infection. InC57BL/6 mice, cerebral malaria was monitored and symptoms classified bythe Rapid Murine Coma and Behavior Scale (50), where 16=no signs,15-11=mild symptomatic, 10-08=prodromal signs of Experimental CerebralMalaria (ECM), 07-04=ECM. Agonal mice were immediately sacrificed.

Administration of IgG from PbMIF immunized mice into naïve BALB/c micethat were infected with PbA provided partial protection, with a delayedrise in parasitemia, a 30% reduction in peak parasitemia, and a 30%prolongation in survival time (FIGS. 10B and C). When studied in theacutely lethal C57BL/6 model of cerebral malaria, IgG from the GFP(control) immunized mice showed no protection: all mice developedneurological signs and became moribund between days 6-7 post-infection(FIGS. 10C and E). By contrast, in C57BL/6 mice that received IgG fromPbMIF immunized mice and then were infected with PbA, there was a markeddelay in the time to develop neurologic disease, less severe disease,and a survival rate of 30%, with complete protection from neurologicmanifestations. Cerebral malaria is a uniformly fatal complication ofPbA infection in C57BL/6 mice and the protective effect ofimmunoglobulin occurred despite equivalent parasitemia between groupsuntil the time of lethality (day 6), after which surviving miceeliminated their parasites (data not shown).

Example 4: Protective Effect of CD4 T Cells from Vaccinated Mice

BALB/c mice (CD45.2⁺) immunized with the RNA/PbMIF-CNE or RNA/GFP-CNEvaccine were infected by i.p. injection of 10⁶ PbA-infected RBCs on day0 and treated with chloroquine on days 7 to 10. At 4 weeks previouslyinfected mice were reinfected and on day 7 post-infection, thesplenocytes were isolated and the CD4 T cells were incubated with 10 mMchloroquine for 2h at 37° C. and CFSE labeled. 5×10⁶ CD4 T cells(CD45.2) then were transferred i.v into naïve CD45.1 BALB/c mice. AllCD45.1 mice were infected with PbA (1×10⁶ iRBCs) at day 3 post-transfer(FIG. 11A). The course of the infection in mice transferred with CD4 Tcells from RNA/GFP-CNE or RNA/PMIF-CNE donors was monitored bydetermining parasitemia by FACS. At day 7 post-infection, mice weresacrificed and CD4⁺CD45.2⁺ donor cells recovered, quantified, andproliferation assessed by CFSE dilution.

Infection was established in recipient mice that received CD4 T cellsfrom the GFP (control) group, as evidenced by increasing parasitemia,but not in mice that received CD4 T cells from the PbMIF immunizeddonors (FIG. 11B).

Parasitemia was significantly reduced in the mice transferred with CD4 Tcells from RNA/PMIF-CNE-immunized donors compared to control micetransferred with CD4 T cells from GFP-CNE-immunized donors. Thus,adoptive transfer of CD4 T cells from PMIF-vaccinated mice alsoconferred protection to parasite re-challenge. In fact, CD4 T cells fromPbMIF immunized mice confer complete protection against blood-stage P.berghei infection.

The protection conferred by the adoptive transfer of CD4 T cells fromthe PbMIF immunized donors was associated with a higher number ofproliferating CD4 T cells (CFSE^(lo)) (FIGS. 11C-E), higher levels ofIFN-γ production (FIG. 11F), and reduced expression of the exhaustionmarker PD-1 (FIG. 11G) when compared to CD4 T cells adoptivelytransferred from the control group.

These data indicate that the augmented CD4 T cell response that developsafter PbMIF immunization in infected mice offers complete protectionagainst infection and is sufficient to prevent the establishment ofblood-stage infection.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

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The invention includes at least the following numbered embodiments:

-   1. A method for providing protective immunity against a parasite    infection in a subject in need thereof, comprising administering an    immunologically effective amount of a composition to the subject,    wherein the composition comprises:    -   (i) a nucleic acid comprising a sequence which encodes a        parasite macrophage migration inhibitory factor (MIF) antigen;    -   (ii) a parasite MIF antigen; or    -   (iii) an antibody which specifically binds to a parasite MIF        antigen.-   2. The method of embodiment 1 wherein the composition comprises an    RNA-based vaccine.-   3. The method of embodiment 1 or 2 wherein the protective immunity    is characterized by protective immunological memory against the    parasite and/or an effective parasite-responsive memory T cell    population.-   4. The method of any preceding embodiment wherein the parasite MIF    antigen comprises a full-length MIF polypeptide or an immunogenic    fragment thereof.-   5. The method of any one of embodiments 1 and 3 wherein the    composition comprises a nucleic acid-based vaccine comprising the    nucleic acid sequence which encodes a parasite MIF antigen.-   6. The method of embodiment 5 wherein the nucleic acid-based vaccine    is an RNA-based vaccine.-   7. The method of embodiment 6 wherein the nucleic acid-based vaccine    comprises a self-replicating RNA molecule.-   8. The method of embodiment 7 wherein the self-replicating RNA is an    alphavirus-derived RNA replicon.-   9. The method of any preceding embodiment wherein the composition    comprises a cationic nano-emulsion (CNE) delivery system.-   10. The method of any one of embodiments 1 to 8 wherein the    composition comprises a lipid nanoparticle (LNP) delivery system.-   11. The method of any preceding embodiment wherein the composition    comprises one or more adjuvants.-   12. The method of embodiment 1, 3 or 4 wherein the antibody which    specifically binds to a parasite MIF antigen comprises polyclonal    antibody.-   13. The method of embodiment 1, 3 or 4 wherein the antibody which    specifically binds to a parasite MIF antigen is a humanized or    chimeric antibody.-   14. The method of any preceding embodiment wherein the parasite is a    parasitic protozoan.-   15. The method of embodiment 14 wherein the protozoan is an    apicomplexan parasite.-   16. The method of embodiment 15 wherein the protozoan belongs to the    genus Plasmodium.-   17. The method of embodiment 14 wherein the protozoan belongs to a    genus selected from the group consisting of: Plasmodium, Toxoplasma,    Babesia, Eimeria, Theileria, Neospora, Sarcocystis, Leishmania, and    Trypanosoma.-   18. The method of any one of embodiments 1 to 13 wherein the    parasite is a parasitic helminth.-   19. The method of embodiment 18 wherein the parasitic helminth is a    nematode.-   20. The method of embodiment 18 or embodiment 19 wherein the    parasitic helminth belongs to a genus selected from the group    consisting of: Ancyclostoma, Necator, Brugia, Wuchereria, Loa,    Mansonella, Trichinella, Trichuris, Ascaris, Anisakis, Dracunculus,    Strongyloides, Haemonchus, Schistosoma and Fasciola.-   21. The method of any preceding embodiment wherein two or more doses    of the composition are administered to the subject.-   22. The method of embodiment 21 wherein the two or more doses are    administered at least 1 week apart, about 2 weeks, about 3 weeks,    about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about    12 weeks, or about 16 weeks apart.-   23. The method of any preceding embodiment wherein the subject is a    vertebrate.-   24. The method of embodiment 23 wherein the subject is a mammal.-   25. The method of embodiment 24 wherein the subject is a human.-   26. The method of embodiment 24 wherein the subject is a veterinary    mammal.-   27. The method of embodiment 26 wherein the veterinary mammal is a    cat, dog, horse, cow, sheep, deer, goat, or pig.-   28. The method of any preceding embodiment wherein the composition    further comprises a nucleic acid sequence which encodes an    additional parasite antigen.-   29. The method of any preceding embodiment wherein the composition    further comprises an additional parasite antigen.-   30. The method of any preceding embodiment wherein the composition    is administered to the subject in combination with a further    composition which comprises a nucleic acid comprising a sequence    which encodes an additional parasite antigen.-   31. The method of any preceding embodiment wherein the composition    is administered to the subject in combination with a further    composition which comprises an additional parasite antigen.-   32. A composition for use in a method of providing protective    immunity against a parasite infection in a subject in need thereof,    which comprises an immunologically effective amount of:    -   (i) a nucleic acid comprising a sequence which encodes a        parasite MIF antigen;    -   (ii) a parasite MIF antigen; or    -   (iii) an antibody which specifically binds to a parasite MIF        antigen.-   33. The composition of embodiment 32 for use in a method according    to any one of embodiments 1 to 31.-   34. A composition comprising an immunologically effective amount of:    -   (i) a nucleic acid comprising a sequence which encodes a        parasite MIF antigen; or    -   (ii) a parasite MIF antigen;    -   wherein the MIF antigen is from a parasitic protozoan.-   35. The composition of embodiment 34 wherein the protozoan is an    apicomplexan parasite, such as Plasmodium.-   36. The composition of embodiment 34 or 35 wherein the protozoan    belongs to a genus selected from the group consisting of:    Plasmodium, Toxoplasma, Babesia, Eimeria, Theileria, Neospora,    Sarcocystis, Leishmania, and Trypanosoma.-   37. A composition comprising an immunologically effective amount of:    -   (i) a nucleic acid comprising a sequence which encodes a        parasite MIF antigen; or    -   (ii) a parasite MIF antigen;    -   wherein the MIF antigen is from a parasitic helminth which        belongs to a genus selected from the group consisting of:        Ancyclostoma, Necator, Brugia, Wuchereria, Loa, Mansonella,        Trichinella, Trichuris, Ascaris, Anisakis, Dracunculus,        Strongyloides, Haemonchus, Schistosoma and Fasciola.-   38. The composition of any one of embodiments 34 to 37 wherein the    parasite MIF antigen comprises a full-length MIF polypeptide or an    immunogenic fragment thereof.-   39. The composition of any one of embodiments 34 to 38 which    comprises a nucleic acid-based vaccine comprising the nucleic acid    sequence which encodes a parasite MIF antigen.-   40. The composition of embodiment 39 wherein the nucleic acid-based    vaccine is an RNA-based vaccine.-   41. The composition of embodiment 40 wherein the nucleic acid-based    vaccine comprises a self-replicating RNA molecule.-   42. The composition of embodiment 41 wherein the self-replicating    RNA is an alphavirus-derived RNA replicon.-   43. The composition of any one of embodiments 34 to 42 which    comprises a cationic nano-emulsion (CNE) delivery system.-   44. The composition of any one of embodiments 34 to 42 which    comprises a lipid nanoparticle (LNP) delivery system.-   45. The composition of any one of embodiments 34 to 44 which    comprises one or more adjuvants.-   46. The composition of any one or embodiments 34 to 45 wherein the    composition further comprises a nucleic acid sequence which encodes    an additional parasite antigen.-   47. The composition of any one or embodiments 34 to 46 wherein the    composition further comprises an additional parasite antigen.-   48. A method of enhancing an immune response to a non-MIF parasite    antigen in a subject, comprising administering an immunologically    effective amount of a composition to the subject, wherein the    composition comprises:    -   (i) a nucleic acid comprising a sequence which encodes a        parasite MIF antigen;    -   (ii) a parasite MIF antigen; or    -   (iii) an antibody which specifically binds to a parasite MIF        antigen.-   49. The method of embodiment 48 which further comprises    administering the non-MIF parasite antigen to the subject.-   50. The method of embodiment 48 which is a method according to any    one of embodiments 1-31.-   51. A method for providing protective immunity against a parasite    infection in a subject in need thereof, comprising administering    parasite-responsive CD4 T cells isolated from a compatible host,    wherein the host has been immunized with a composition comprising:    -   (i) a nucleic acid comprising a sequence which encodes a        parasite MIF antigen; or    -   (ii) a parasite MIF antigen.-   52. The method of embodiment 51 wherein the compatible host has been    administered a composition according to a method of any one of    embodiments 1-31.

1. A composition for use in a method of providing protective immunityagainst a parasite infection in a subject in need thereof, whichcomprises an immunologically effective amount of one or more of: (i) anucleic acid comprising a sequence which encodes a parasite macrophagemigration inhibitory factor (MIF) antigen; (ii) a parasite MIF antigen;or (iii) an antibody which specifically binds to a parasite MIF antigen.2. The composition for use according to claim 1 (i) wherein thecomposition comprises an RNA-based vaccine.
 3. The composition for useaccording to claim 1 or claim 2 wherein the protective immunity ischaracterized by protective immunological memory against the parasiteand/or an effective parasite-responsive memory T cell population.
 4. Thecomposition for use according to claim 1, 2 or 3 wherein the compositioncomprises a nucleic acid-based vaccine comprising the nucleic acidsequence which encodes a parasite MIF antigen; for example, wherein thenucleic acid-based vaccine is a RNA-based vaccine, which may comprises aself-replicating RNA molecule, such as an alphavirus-derived RNAreplicon.
 5. The composition for use according to any preceding claimwherein the composition comprises a cationic nano-emulsion (CNE)delivery system or a lipid nanoparticle (LNP) delivery system.
 6. Thecomposition for use according to any preceding claim wherein thecomposition comprises one or more adjuvants.
 7. The composition for useaccording to claim 1 wherein the antibody which specifically binds to aparasite MIF antigen comprises monoclonal antibody or polyclonalantibody.
 8. The composition for use according to claim 1 wherein theantibody which specifically binds to a parasite MIF antigen is a human,humanized or chimeric monoclonal antibody.
 9. The composition for useaccording to any preceding claim wherein the parasite is a parasiticprotozoan, for example wherein (i) the protozoan is an apicomplexanparasite; and/or (ii) the protozoan belongs to a genus selected from thegroup consisting of: Plasmodium, Toxoplasma, Babesia, Eimeria,Theileria, Neospora, Sarcocystis, Leishmania, and Trypanosoma.
 10. Thecomposition for use according to any one of claims 1 to 8 wherein theparasite is a parasitic helminth, such as a nematode.
 11. Thecomposition for use according to claim 10 wherein the parasitic helminthbelongs to a genus selected from the group consisting of: Ancyclostoma,Necator, Brugia, Wuchereria, Loa, Mansonella, Trichinella, Trichuris,Ascaris, Anisakis, Dracunculus, Strongyloides, Haemonchus, Schistosomaand Fasciola.
 12. The composition for use according to any precedingclaim wherein the subject is a vertebrate, such as a mammal e.g. a humanor a veterinary mammal.
 13. The composition for use according to anypreceding claim wherein: (a) the composition further comprises a nucleicacid sequence which encodes an additional parasite antigen, and/or (b)the composition further comprises an additional parasite antigen, and/or(c) the composition is administered to the subject in combination with afurther composition which comprises a nucleic acid comprising a sequencewhich encodes an additional parasite antigen; and/or (d) the compositionis administered to the subject in combination with a further compositionwhich comprises an additional parasite antigen.
 14. A compositioncomprising an immunologically effective amount of: (i) a nucleic acidcomprising a sequence which encodes a parasite MIF antigen; or (ii) aparasite MIF antigen; wherein the MIF antigen is from: (a) a parasiticprotozoan; or (b) a parasitic helminth which belongs to a genus selectedfrom the group consisting of: Ancyclostoma, Necator, Brugia, Wuchereria,Loa, Mansonella, Trichinella, Trichuris, Ascaris, Anisakis, Dracunculus,Strongyloides, Haemonchus, Schistosoma and Fasciola.
 15. The compositionof claim 14 wherein the parasitic protozoan is an apicomplexan parasiteand/or belongs to a genus selected from the group consisting of:Plasmodium, Toxoplasma, Babesia, Eimeria, Theileria, Neospora,Sarcocystis, Leishmania, and Trypanosoma.
 16. The composition of any oneof claims 14 to 15 wherein the parasite MIF antigen is a full-length MIFpolypeptide or an immunogenic fragment thereof.
 17. The composition ofany one of claims 14 to 16 which comprises a nucleic acid-based vaccinecomprising the nucleic acid sequence which encodes a parasite MIFantigen; for example, wherein the nucleic acid-based vaccine is aRNA-based vaccine, such as a self-replicating RNA molecule, which may bean alphavirus-derived RNA replicon.
 18. The composition of any one ofclaims 14 to 17 which comprises a cationic nano-emulsion (CNE) deliverysystem or a lipid nanoparticle (LNP) delivery system.